Novel chimeric ligand-gated ion channels and methods of use thereof

ABSTRACT

The present invention provides novel chimeric receptors that have unique pharmacology. In particular, the chimeric receptors comprise a mutated ligand binding domain of the α7 nicotinic acetylcholine receptor fused to a transmembrane or channel domain from a ligand-gated ion channel protein. The mutations in the ligand binding domain confer selective binding of compounds. Methods of using the novel chimeric receptors of the invention as well as compounds that preferentially bind and activate the chimeric receptors are also disclosed.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.61/136,856, filed Oct. 9, 2008, which is herein incorporated byreference in its entirety.

DESCRIPTION OF THE TEXT FILE SUBMITTED ELECTRONICALLY

The contents of the text file submitted electronically herewith areincorporated herein by reference in their entirety: A computer readableformat copy of the Sequence Listing (filename:HOWA_(—)003_(—)01US_SeqList_ST25.txt, date recorded: Oct. 9, 2009, filesize 51 kilobytes).

FIELD OF THE INVENTION

The present invention relates to novel chimeric ligand-gated ionchannels having alterations in their ligand binding domains that conferpharmacological selectivity for novel small molecule synthetic ligands.Methods of using these novel chimeric ligand-gated ion channels tomodulate neuronal activity as well as therapeutic indications for thechimeric receptors are also disclosed.

BACKGROUND OF THE INVENTION

Ligand-gated ion channels (LGICs) transduce chemical signals intoelectrical activity by increasing the permeability of neurons tospecific ions, resulting in current flow, which alters the propensity ofneurons to fire action potentials. Action potentials are rapidfluctuations in neuronal voltage that are used for communicatinginformation in the nervous system. Because many neurological disordersare related to neural activity (e.g. pain and epilepsy), ion channels,including LGICs, have been targeted pharmacologically for clinicaltherapeutics (Brunton et al. (2006) Goodman & Gilman's ThePharmacological Basis of Therapeutics).

Neuronal activity can be enhanced by administering an agonist for acation-selective (excitatory) LGIC (e.g. glutamate receptor) or neuronalactivity can be suppressed or silenced by administering an agonist foran anion-selective (inhibitory) LGIC (e.g. GABA receptor). In order tomodulate neuronal activity of a subset of neurons in the nervous systemfor a therapeutic effect, these small molecule ligands for LGICs wouldrequire targeting by injection, iontophoresis, or reverse dialysis to avolume of the neuropil. However, these techniques are invasive,especially for targets deep in the brain. An even greater limitation isthat these perturbations within the targeted brain region are not celltype-specific due to the widespread presence of LGICs (e.g. glutamateand GABA ion channels) on nearly all neurons. Perturbation of neuronsthat are not involved in the therapeutic effect can lead to significantundesired side effects.

Thus, there is a need in the art to develop LGICs that can beselectively activated with tailored compound ligands. Such novel LGICs,once delivered to the neurons of interest by gene therapy methods, wouldrender these neurons sensitive to a ligand selective for such novelLGICs and would obviate the need for local delivery of the ligand, sincethe tailored ligand would have no effect on native LGICs. Furthermore,selective activation of these novel LGICs would eliminate thenon-specific effects arising from activation of neighboring populationsof neurons that inevitably occur due to the ubiquitous expression ofnative LGICs. This would provide specificity for control of neuronactivity that could be used therapeutically to treat diseases such asepilepsy and chronic pain. Also, by manipulating activity of neuronpopulations that control hunger and satiety, these LGICs and associatedligands could also be used to treat diseases associated with undesiredbehaviors such as overeating or anorexia. Therefore, development ofnovel LGICs with unique pharmacology would have therapeutic utility.

SUMMARY OF THE INVENTION

The present invention provides novel chimeric receptors that can beselectively activated by compounds. In one embodiment, the chimericreceptor comprises a ligand binding domain from an α7 nicotinicacetylcholine receptor fused to a transmembrane domain from aligand-gated ion channel protein, wherein the ligand binding domaincomprises at least one mutation that confers selective binding to acompound. The transmembrane domain may be from a ligand-gated ionchannel from the Cys-loop family of receptors. In some embodiments, thechimeric receptor comprises a transmembrane domain from a cationselective channel, such as the 5HT3 receptor. In other embodiments, thechimeric receptor comprises a transmembrane domain from an anionselective channel, such as the glycine receptor or the GABA C receptor.

The ligand binding domain of the chimeric receptor comprises at leastone mutation that confers a unique pharmacology to the chimericreceptor. In some embodiments, the ligand binding domain of the α7nicotinic acetylcholine receptor contains a point mutation at positions77, 79, or 141 in SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 10. Themutations may include Q79A, Q79G, L141A, L141F, L141P, W77F, W77Y, orW77M substitutions in SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 10.

The present invention also includes compounds that preferentially bindand activate the mutated chimeric receptors of the invention. In oneembodiment, the compound has the structure of formula I:

or a pharmaceutically acceptable salt thereof, wherein A, R₁, R₂, R₃, R₄and R₅ are as defined below.

In another embodiment, the compound has the core structure of formulaII:

or a pharmaceutically acceptable salt thereof, wherein A, R^(A), R^(B)and n are as defined below.

In another embodiment, the compound has the core structure of formulaIII:

wherein each

is a single or double bond, and R^(A), R^(B), R^(C), R^(D) and E are asdefined below.

The present invention also provides a method of modulating theexcitability of a neuronal cell. In one embodiment, the method comprisesexpressing in the neuronal cell a genetic construct encoding a chimericreceptor, wherein the chimeric receptor comprises a ligand bindingdomain from an α7 nicotinic acetylcholine receptor fused to atransmembrane domain from a ligand-gated ion channel protein, whereinsaid ligand binding domain comprises at least one mutation that confersselective binding to a compound of formula I, II or III as describedherein; and exposing the neuronal cell to the compound. In anotherembodiment, the excitability of the neuron is increased. In stillanother embodiment, the excitability of the neuron is decreased. Theneuronal cell may be in vitro or in vivo.

The present invention also encompasses kits comprising chimericreceptors of the invention as disclosed herein and tailored compoundligands. In some embodiments, the chimeric receptor comprises a ligandbinding domain from an α7 nicotinic acetylcholine receptor fused to atransmembrane domain from a 5HT3 receptor, wherein said ligand bindingdomain comprises at least one mutation that confers selective binding toa compound of formula I, II or III as described herein. In otherembodiments, the chimeric receptor comprises a ligand binding domainfrom an α7 nicotinic acetylcholine receptor fused to a transmembranedomain from a glycine receptor or the GABA C receptor, wherein saidligand binding domain comprises at least one mutation that confersselective binding to a compound of formula I, II or III as describedherein. At least one mutation may include W77F, Q79A, Q79G, L141F, orL141P substitutions in SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 10.

The present invention also provides a method of treating a disease ordisorder associated with the nervous system in a subject in needthereof. In one embodiment, the method comprises delivering a geneticconstruct to a population of neurons in the subject, wherein the geneticconstruct encodes a mutant α7 nicotinic acetylcholine receptor, whereinthe mutation confers selective binding to a compound of formula I, II orIII as described herein; and administering the compound to the subject.In some embodiments, the receptor is a chimeric receptor, wherein thechimeric receptor comprises a ligand binding domain from an α7 nicotinicacetylcholine receptor fused to a transmembrane domain from aligand-gated ion channel protein, wherein said ligand binding domaincomprises at least one mutation that confers selective binding to acompound of formula I, II or III as described herein. In someembodiments, the activity of the population of neurons is increasedfollowing administration of the compound to the subject. In otherembodiments, the activity of the population of neurons is decreasedfollowing administration of the compound to the subject. In certainembodiments, the disorder to be treated is epilepsy or chronic pain.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1. “Bump-hole” approach to engineering selective ion channel-ligandinteractions. A. Design scheme for mutant ion channels that selectivelybind a synthetic ligand, wherein the synthetic ligand cannot bind to thenatural, endogenous channel. B. Chemical structures of acetylcholine(ACh), nicotine, and PNU-282987. C. Homology model of the human α7 nAChRligand binding domain (LBD) with docked agonist, PNU-292897, based onthe x-ray crystal structure of the snail AChBP showing proximity to theamino acids W77, Q79, Q139, and L141. These residues were targeted formutagenesis based on their proximity to the benzamide functionality ofPNU-282987 in this model. D. Ionic current evoked by PNU-282987 appliedto an human embryonic kidney cell expressing the chimeric ion channelα7-5HT3. A large peak current (I_(peak)) rapidly decays to a persistent,smaller current (I_(SS)). E. Fluoresence change associated with a highthroughput membrane potential (MP) assay that was used to screen mutantion channels against potential ligands. MP response was persistent overseveral minutes. F. Dose responses show that response from MP assaycorresponds to I_(SS). G. Colonmap showing the relative activity ofα7-5HT3 mutants. Responses to ACh (100 μM), nicotine (100 μM), andPNU-282987 (10 μM) were normalized to the response to ACh (100 μM). Therelative amount of cell surface α-Bgt binding sites was measured bynormalizing the fluorescent intensity of α-Bgt-594 bound to α7-5HT3mutants relative to cells expressing α7-5HT3 under non-permeablizingconditions.

FIG. 2. Components of the quinclidine benzamide library used to identifyligands selective for mutant ligand binding domains. Top left in boldshows the (R)- and (S)-enantiomers of 3-aminoquinulidine that werecoupled to the displayed benzoic acid derivatives. Wavy lines indicatejoining point to form the quinclidine benzamides. R or S in numberingscheme refers to which enantiomers were synthesized.

FIG. 3. Colormap of EC50s from dose response curves of α7-5HT3 variantsvs. ACh, nicotine, and aminoquinuclidine benzamides. In order tohighlight molecules that are highly selective for α7-5HT3 mutantchimeric receptors vs. the α7-5HT3 wild-type chimeric receptor, the datahas been sorted such that EC50 values for molecules with EC50<30 μM forthe wild-type α7-5HT3 chimeric receptor (top row) are not shown forα7-5HT3 mutant chimeric receptors.

FIG. 4. Examples of dose responses showing selective interactionsbetween ligands and mutated ligated-gated ion channels. Dose responsecurves for some compounds with EC50s (shown in parentheses) less than 10μM against mutant ligand-gated ion channels showing negligibleactivation of the α7-5HT3 chimeric channel with non-mutated or“wild-type” (wt) sequence.

FIG. 5. Dose response curves for mutant ligand-gated ion channel-ligandinteractions that show orthogonal pharmacology, indicating that theligand-ion channel systems can be used in the presence of one anotherwith minimal crosstalk.

FIG. 6. Dose response curves for compounds 22S and 38R on the wild-typeα7-5HT3 (open circles), single mutant Q79G α7-5HT3 (filled blackcircles), and double mutant Q79G, Q139M α7-5HT3 (filled red circles)chimeric receptors.

FIG. 7. High conductance chimeric cationic ligand-gated ion channelactivates layer ⅔ cortical neurons. A. Current trace from voltage clamprecordings in HEK 293 cells transfected with high conductance (HC)α7-5HT3 L141F receptors showing sustained response of the receptor tocompound 89S. B. Cell attached extracellular recording of actioncurrents from mouse layer ⅔ cortical neurons transfected with HC α7-5HT3L141F. In addition to the mutation in the ligand binding domain (e.g.,L141F), these receptors contain high conductance inducing mutations inthe α7-5HT3 sequence: R425Q R429D R433A in SEQ ID NO: 1.

FIG. 8. Chimeric α7-GlyR ligand-gated ion channel conducts chloride andgreatly reduces excitability of cortical neurons in the presence of thecognate ligand. A. Current trace from voltage clamp recordings intransfected HEK 293 cells showing sustained responses of α7-GlyR andα7-GABA C mutant chimeric receptors to activation by synthetic ligandsof the invention. Black line indicates time course of ligandapplication. B. I-V trace showing that the reversal potential (V_(rev))of the drug-induced conductance in the chimeric mutant ligand-gated ionchannel α7-GlyR L141F is identical to the chloride reversal potential(E_(Cl)), indicating that the channel is chloride selective. C.Rheobase, which is the amount of current required to elicit an actionpotential, is not affected by expression of α7-GlyR L141F Y115F (labeledas condition: PRE). However, rheobase increases 10-fold in the presenceof 10 μM compound 89S. There is no change in rheobase in untransfectedneurons, even in the presence of 30 μM compound 89S. This reduction ofexcitability in α7-GlyR L141F Y115F expressing neurons is reversed afterwashout of the synthetic ligand (labeled as condition: WASH).

FIG. 9. Synthetic ligand-induced silencing of a cortical neurontransduced by viral gene delivery α7-GlyR L141F mutant chimericligand-gated ion channels. Cortical neurons were transduced withadeno-associated virus (AAV) encoding the α7-GlyR L141F mutant chimericligand-gated ion channel under the control of the neuron-specificsynapsin promoter. A. Excitability of a transfected cortical neuronbefore, during, and after administration of compound 89S (10 μM). Evenafter injection of current amplitude up to 500 pA, the neuron is poorlyexcitable, firing only a few action potentials in the presence ofCompound 89S. The neuron fires normally with <100 pA injected currentbefore and after the compound administration. B. Excitability of anon-transduced neuron is shown for comparison. Non-transduced neuronsare not affected by administration of compound 89S, even at a higherconcentration (30 μM).

FIG. 10. Amino acid sequence of the wild-type α7-5HT3 chimeric receptor(SEQ ID NO:1).

FIG. 11. Amino acid sequence of the wild-type α7-GlyR chimeric receptor(SEQ ID NO: 6).

FIG. 12. Amino acid sequence of the wild-type α7-GABA C chimericreceptor (SEQ ID NO: 10).

FIG. 13. Synthetic ligands for mutant chimeric α7-5HT3 receptors. TheEC50 for each compound for wild-type (WT) and two of the mutant chimericreceptors (Q79G and L141F) are shown below each compound.

DETAILED DESCRIPTION OF THE INVENTION

Manipulation of electrical activity in neurons provides a powerfulapproach to regulate physiology and behavior. Effective methods formodulating neuronal activity in vivo should provide: 1) rapid onset ofperturbation (seconds to minutes); 2) an ion channel that isnon-perturbative to targeted cell types under basal conditions; 3)ligands selective for the ion channel targeted to defined cell types; 4)a straightforward, single transgene genetic strategy without need tointerfere with endogenous ion channels, and 5) the capability toindependently and orthogonally perturb multiple populations. Severalapproaches derived from invertebrate (Slimko et al. (2002) J. Neurosci.,Vol. 22: 7373-7379) or vertebrate LGICs (Arenkiel et al. (2008) NatureMethods, Vol. 5: 299-302) and G-protein coupled receptors (Armbruster etal. (2007) Proc. Natl. Acad. Sci. USA, Vol. 104: 5163-5168) do notsatisfy all of these requirements (Luo et al. (2008) Neuron, Vol. 57:634-660). A major challenge in overcoming the limitations of thesepreviously developed systems is the paucity of knowledge about therelationship of structure to function in most of the ion channel orreceptor systems in use today.

To overcome the limitations of traditional methods of neuronalactivation, transgenic and gene therapy strategies can be used to targetnovel ion channels with unique pharmacology to specific cellpopulations. Transgenic and gene therapy strategies use celltype-selective promoter activity to target gene expression of the novelion channels to cell types. The present invention is based, in part, onthe discovery that mutations in the ligand binding domain (LBD) of theα7 nicotinic acetylcholine receptor (α7 nAChR) can conferpharmacological specificity such that compound ligands can be tailoredto bind uniquely to the mutant receptor. These receptors having uniquepharmacology can be used in combination with their tailored compoundligands to modulate the activity of specific neuronal populations.

The nicotinic acetylcholine receptor is probably the best understoodmember of the Cys-loop family of ligand-gated ion channels (LGICs) afterover 30 years of structure-function analysis. In the picture that hasemerged, the residues in the binding pocket of the α7 nAChR LBD thatcontribute to channel activation have been described to involve anaromatic cage consisting of multiple tyrosine and tryptophan residues(Galzi et al. (1991) FEBS Letters, Vol. 294: 198-202). This analysis wasconfirmed by an x-ray crystal structure of the homologous acetylcholinebinding protein (AChBP), which provides a more complete structuralrationale for ligand binding (Celie et al. (2004) Neuron, Vol. 41:907-914). Furthermore, the α7 nAChR LBD can be spliced to thetransmembrane domain of the cationic serotonin receptor 3a (α7-5HT3) orthe chloride-selective glycine receptor (α7-GlyR1) to generate chimericreceptors (Eisele et al. (1993) Nature, Vol. 366: 479-483; Grutter etal. (2005) Proc. Natl. Acad. Sci. USA, Vol. 102: 18207-18212). Thistransferability of the pharmacologic element (e.g., LBD) to multiple ionconducting elements (channel or transmembrane domains) is a usefulfoundation for optimizing function so that channels have the preferredproperties. The major challenge in using ion channels with native ligandbinding domains and their corresponding ligands as tools to manipulateneuronal activity is that these native ligand binding domains arealready found in the brain, and thus the small molecule ligands willperturb electrical activity in multiple undesired cell populations.

To overcome these issues, the present invention describes an approach tomodify the ligand recognition properties of the nAChR using a“bump-hole” strategy (FIG. 1A). These modified ligand binding domains(LBDs) of the α7 nAChR are modular units that can be combined withchannel domains (e.g. transmembrane domains) to generate chimericreceptors with desired ligand selectivity and conductance properties.Accordingly, the present invention provides chimeric receptorscomprising a ligand binding domain from an α7 nicotinic acetylcholinereceptor fused to a transmembrane domain from a ligand-gated ion channelprotein, wherein the ligand binding domain comprises at least onemutation that confers selective binding to a compound. A “chimericreceptor” refers to a receptor comprising at least one domain from afirst protein and at least one domain from a second protein. The firstand second proteins may be from the same species (i.e. both humanproteins) or may be from different species (i.e. one human protein andone mouse protein).

As used herein, the term “ligand binding domain” refers to theextracellular region of a protein receptor that interacts with acompound such that a conformational change of the region occurs uponbinding of the compound. The conformational change of the ligand bindingdomain typically produces activation of the receptor. In the case ofligand-gated ion channels, binding of the ligand to the ligand bindingdomain opens the ion channel. As used herein, the term “transmembranedomain” is used interchangeably with “channel domain” or “ion poredomain” (IPD) and refers to the region of the protein receptor thatspans the lipid membrane of the cell and forms the channel or pore inthe membrane through which ions can pass between the extracellularmilieu and the cellular cytoplasm. The ligand binding domain of the α7nAChR is functionally fused to a channel or transmembrane domain from aligand-gated ion channel. “Functionally fused” means that the twoprotein domains are linked such that binding of a ligand to the ligandbinding domain will result in a conformational change that opens the ionchannel (i.e. increases channel conductance).

In one embodiment, the transmembrane domain is from a ligand-gated ionchannel of the Cys-loop family of ionotropic receptors. Examples ofligand-gated ion channels from this family include, but are not limitedto, ionotropic nicotinic acetylcholine receptors, ionotropic serotoninreceptors (e.g. 5HT3), ionotropic glycine receptors, and ionotropic GABAreceptors (e.g. GABA_(A) and GABA_(C) receptors). In some embodiments,the ligand-gated ion channels are selective for cations, such as the5HT3 and nicotinic acetylcholine receptors. In other embodiments, theligand-gated ion channels are selective for anions, such as the GABA andglycine receptors. In preferred embodiments, the transmembrane domain ofthe chimeric receptor is the transmembrane domain of a 5HT3, nAChR,glycine, or GABA C receptor.

In another embodiment of the invention, there is at least one mutationin the ligand binding domain of the α7 nAChR that confers selectivebinding to a compound (e.g., selectivity-inducing mutation). Themutation can include a point mutation in the amino acid residue inposition 77, 79, 139, or 141 in the amino acid sequence listed in SEQ IDNO: 1, SEQ ID NO: 6, or SEQ ID NO: 10. Non-limiting examples of thepoint mutations include Q79A, Q79G, L141A, L141F, L141P, W77F, W77Y, andW77M, where the identity of the amino acids is designated by the singleletter amino acid code. Other suitable amino acid point mutations thatalso confer selectivity are shown in FIG. 3 and include Q79C, Q79D,Q79E, Q79H, Q79L, Q79P, Q79R, Q79S, Q79T, Q79W, Q139A, Q139C, Q139D,Q139F, Q139G, Q139H, Q1391, Q139K, Q139L, Q139M, Q139N, Q139R, Q139S,Q139V, Q139W, Q139Y, L141G, L141H, L141I, L141M, L141N, L141Q, L141S,L141V, and L141W. In a preferred embodiment, the at least one mutationin the ligand binding domain is W77F in SEQ ID NO: 1, SEQ ID NO: 6, orSEQ ID NO: 10. In another preferred embodiment, the at least onemutation in the ligand binding domain is Q79A or Q79G in SEQ ID NO: 1,SEQ ID NO: 6, or SEQ ID NO: 10. In still another preferred embodiment,the at least one mutation in the ligand binding domain is L141F or L141Pin SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 10. In some embodiments,the chimeric receptor has the amino acid sequence of SEQ ID NO: 2, SEQID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ IDNO: 9, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO: 13. The ligandbinding domain of the chimeric receptor may have more than one mutationin the amino acid sequence. By way of example, the ligand binding domainof the α7 nAChR may comprise a point mutation at position 79 and 141 ofthe amino acid sequence or at position 79 and 139 of the amino acidsequence. Any of the specific mutations disclosed above as well asadditional mutations may be made in combination to generate double,triple, or multiple mutant chimeric receptors.

The receptors of the invention can also contain mutations in the ligandbinding domain of the α7 nAChR that significantly reduces responsivenessto the naturally occurring ligand, acetylcholine. Because acetylcholineis produced in the brain, mutations that limit the sensitivity ofchimeric channels to ACh reduce the possibility of the channel beingactivated in the absence of the synthetic ligand. Suitable pointmutations that reduce binding to acetylcholine include Y115F, Q79R,Q139G, Q139V, Q139W, Q139Y, L141A, L141Q, L141S, with Y115F, Q139G,L141A, and L141S being preferred in some embodiments. These mutationscan be combined with any of the selectivity-inducing mutations asdescribed herein, such as Q79G and L141F.

In some embodiments, the receptors of the invention also have mutationsin the transmembrane domain, specifically the M2 region and regionsflanking the M2 region of Cys-loop ion channels, which affectconductance and desensitization properties.

In other embodiments, in addition to the selectivity-inducing mutationsdescribed herein, the receptors of the invention have mutations in thecytoplasmic domains, specifically in the M3-M4 loop, which affect ionconductance properties. For instance, for chimeric receptors containingthe 5HT3 transmembrane domain, deletion of portions of the M3-M4 loop,replacement with sequences from other Cys-loop receptors, or specificmutations such as the triple mutation: R425Q R429D R433A modulate theconductance of the ion channel. In one embodiment, in addition to atleast one selectivity-inducing mutation, the chimeric receptor containsthe triple mutation R425Q R429D R433A in SEQ ID NO: 1, which increasesconductance in α7-5HT3 chimeric receptors (referred to as highconductance or HC).

The present invention also encompasses nucleic acids encoding the mutantchimeric receptors of the invention. In some embodiments, the nucleicacids encode a mutant chimeric receptor having an amino acid sequence ofSEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 7,SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ ID NO: 12, or SEQ ID NO:13. In one embodiment, the nucleic acid encodes a chimeric receptorcomprising the ligand binding domain of the α7-nAChR and an ion poredomain from the GABA C (SEQ ID NO: 10). The nucleic acids encoding thechimeric receptors may be operably linked to a promoter and incorporatedinto a genetic construct or vector. The promoter may be an induciblepromoter and/or a tissue specific promoter. For example, neuron-specificpromoters such as synapsin, CAMKII, and neuron-specific enolase can beused to target neurons selectively over interspersed cell classes suchas glia and epithelia cells. TRPV1 promoter can be used to targetnociceptive sensory neurons which give rise to the pain transmittingC-fibers. In addition, POMC, NPY, AGRP, MCH, and Orexin promoters can beused to target neurons involved in obesity or anorexia. Other suitablepromoters can be ascertained by one of skill in the art depending on theparticular population of cells to be targeted. The nucleic acid encodingthe chimeric receptor may be incorporated into the genome of a cell orit may be contained within a vector. The term “vector” refers to themeans by which a nucleic acid can be propagated and/or transferredbetween organisms, cells, or cellular components. Vectors includeplasmids, viruses, bacteriophages, pro-viruses, phagemids, transposons,artificial chromosomes, and the like, that replicate autonomously or canintegrate into a chromosome of a host cell. A vector can also be a nakedRNA polynucleotide, a naked DNA polynucleotide, a polynucleotidecomposed of both DNA and RNA within the same strand, apoly-lysine-conjugated DNA or RNA, a peptide-conjugated DNA or RNA, aliposome-conjugated DNA, or the like, that is not autonomouslyreplicating. In some embodiments, the nucleic acid encoding the chimericreceptor is contained within a plasmid or viral vector.

General texts which describe molecular biological techniques, which areapplicable to the present invention, such as cloning, mutation, and thelike, include Berger and Kimmel, Guide to Molecular Cloning Techniques,Methods in Enzymology volume 152 Academic Press, Inc., San Diego, Calif.(Berger); Sambrook et al., Molecular Cloning—A Laboratory Manual (3rdEd.), Vol. 1-3, Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.,2000 (“Sambrook”) and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., (“Ausubel”).These texts describe mutagenesis, the use of vectors, promoters and manyother relevant topics related to, e.g., the production of the novelchimeric receptors of the invention. Methods of protein engineering andrecombinant DNA technology are known to those of skill in the art andcan be employed to produce the chimeric receptors of the invention giventhe guidance described herein.

The present invention also includes a method of modulating theexcitability of a neuronal cell. “Excitability” refers to the ability ofneurons to generate and propagate action potentials. Increasedexcitability results in a lower threshold for action potentialgeneration (i.e. less current is required to trigger an actionpotential), while decreased excitability increases the threshold foraction potential generation (i.e. more current is required to trigger anaction potential). In one embodiment, the method comprises expressing inthe neuronal cell a genetic construct encoding a chimeric receptor,wherein the chimeric receptor comprises a ligand binding domain from anα7 nicotinic acetylcholine receptor fused to a transmembrane domain froma ligand-gated ion channel protein, wherein said ligand binding domaincomprises at least one mutation that confers selective binding to acompound; and exposing the neuronal cell to the compound. The neuronalcell may be in vitro or in vivo.

Any of the novel chimeric receptors described herein may be used inmethods of modulating neuronal excitability. In one embodiment, thechimeric receptor comprises a transmembrane domain from a ligand-gatedion channel protein that is selective for cations. In a preferredembodiment, the transmembrane domain is a transmembrane domain from the5HT3 receptor (α7-5HT3). Activation of these “cationic” types ofchimeric receptors with their compound ligands can increase theexcitability of neuronal cells expressing such chimeric receptors.

In another embodiment, the chimeric receptor comprises a transmembranedomain from a ligand-gated ion channel protein that is selective foranions. In a preferred embodiment, the transmembrane domain is atransmembrane domain from the glycine receptor (α7-GlyR1). In a anotherpreferred embodiment, the transmembrane domain is a transmembrane domainfrom the GABA C receptor (α7-GABA C). Activation of these “anionic”types of chimeric receptors with their compound ligands can decrease theexcitability of neuronal cells expressing such chimeric receptors.

The chimeric receptors comprise a ligand binding domain from the α7nicotinic acetylcholine receptor that has at least one mutation thatconfers selective binding to a compound described herein. In someembodiments, the at least one mutation is selected from the groupconsisting of Q79A, Q79G, L141A, L141F, L141P, W77F, W77Y, and W77M inSEQ ID NO: 1. In other embodiments, the at least one mutation isselected from the group consisting of Q79A, Q79G, L141A, L141F, L141P,W77F, W77Y, and W77M in SEQ ID NO: 6. In certain embodiments, the atleast one mutation is selected from the group consisting of Q79A, Q79G,L141A, L141F, L141P, W77F, W77Y, and W77M in SEQ ID NO: 10. Preferably,the compound does not activate a wild-type α7 nicotinic acetylcholinereceptor.

The compounds described herein can be used in conjunction with the novelchimeric receptors in: (a) the methods of modulating neuronalexcitability as described herein; (b) the kits of the present inventionas described herein; and (c) the methods for treating a disease ordisorder of the nervous system as described herein. In one embodiment,the compound is selected from the group consisting of Compound Nos. 3R,6R, 9S, 12R, 12S, 14S, 16S, 19R, 19S, 21S, 22S, 28S, 34S, 35S, 37S, 38R,39S, 40S, 41R, 41S, 42R, 42S, 85S, 86S, 88S, 89S, 90S, 91S, 96R, 97R,115S, 117S, 118S, 119S, 120S, 121S, 127S, 131S, 132S, 134S, 148S, 149S,154S, 156S, 157S, 158S, 163S, 164S, 165S, 170R, 208S, 212, 241, 242,245, 253, 254, 255, 278S, 279S, 281S, 292R, 294S, 295S, and 296S.

In some embodiments, a particular chimeric receptor may be used with oneor more particular compounds. Non-limiting examples of some of thesecombinations include: (1) a W77F α7-5HT3 (SEQ ID NO: 2), W77F α7-GlyR1(SEQ ID NO: 7), or W77F α7-GABA C (SEQ ID NO: 11) chimeric receptor withthe 28S, 34S, 96R, 97R, 131S, 132S, 278S, 279S, or 281S syntheticcompound; (2) Q79A α7-5HT3, Q79G α7-5HT3 (SEQ ID NO: 3), Q79A α7-GlyR1,Q79G α7-GlyR1 (SEQ ID NO: 8), Q79A α7-GABA C, or Q79G α7-GABA C (SEQ IDNO: 12) chimeric receptor with the 9S, 16S, 22S, 38R, 115S, 117S, 134S,148S, 149S, 154S, 156S, 157S, 158S, 163S, 164S, 165S, 170R, 292R, 295S,or 296S synthetic compound; and (3) L141F α7-5HT3 (SEQ ID NO: 4), L141Pα7-5HT3, L141F α7-GlyR1 (SEQ ID NO: 9), or L141P α7-GlyR1, L141F α7-GABAC (SEQ ID NO: 13), or L141P α7-GABA C chimeric receptor with any one ofCompound Nos. 19S, 85S, 86S, 88S, 89S, 90S, 91S, 1185, 119S, 120S, 121S,127S, 208S, 212, 241, 242, 245, 253, 254, 255, or 2945.

Efficacy of a synthetic ligand in activating a particular mutantchimeric receptor can be measured by one of several assays including,but not limited to, a fluorescence membrane potential assay, radioactivebinding assays, and voltage clamp measurement of peak currents andsustained currents (see, e.g., Examples 2 and 4). For instance, changesin ion flux may be assessed by determining changes in electricalpotential of the cell or membrane expressing a novel chimeric receptorof the invention upon exposure to a particular synthetic compound.Changes in current and membrane potential can be measured withvoltage-clamp and patch-clamp techniques as known in the art. Otherknown assays include radiolabeled ion flux assays and fluorescenceassays using voltage-sensitive dyes (see, e.g., Vestergarrd-Bogind etal., J. Membrane Biol., 88: 67-75 (1988); Gonzales & Tsien, Chem. Biol.,4: 269-277 (1997); Daniel et al., J. Pharmacol. Meth., 25: 185-193(1991); Holevinsky et al., J. Membrane Biology, 137: 59-70 (1994)).Thus, the invention encompasses synthetic ligands that exhibit anEC50>70 μM, 75 μM, 80 μM, 85 μM, 90 μM, 95 μM, or >100 μM against thewild-type α7 nAChR and an EC50<15 μM, 12 μM, 10 μM, 8 μM, 6 μM, 5 μM, 4μM, 2 μM, or <1 μM against a mutant α7 receptor as measured by any ofthe above-described assays (e.g., fluorescence membrane potential assayor peak/sustained current assay). An “EC50” or “half maximal effectiveconcentration” as used herein is the concentration at which thesynthetic ligand produces a half-maximal response. Synthetic ligandsthat produce >40%, 45%, 50%, 55%, 60%, 65%, 70%, or >75% maximalresponse on mutant receptors in a HEK cell membrane potential assay (seeExample 2) relative to the agonist response on a wild-type receptor to afull agonist such as acetylcholine are also included in the invention.

There are a number of methods known in the art for introducing thegenetic construct encoding the novel chimeric receptors of the inventionin the cells of interest (e.g., neuronal cells). For example, the vectoror genetic construct can be transferred into a host cell by physical,chemical or biological means. Physical methods for introducing apolynucleotide into a host cell include calcium phosphate precipitation,DEAE-dextran, lipofection, particle bombardment, microinjection,electroporation, cell sonication, receptor-mediated transfection, andthe like.

Biological methods for introducing a polynucleotide of interest into ahost cell include the use of DNA and RNA vectors. Viral vectors, andespecially retroviral vectors, have become the most widely used methodfor inserting genes into mammalian, e.g., human cells. Other viralvectors can be derived from lentivirus, poxviruses, herpes simplex virusI, adenoviruses and adeno-associated viruses, vaccinia virus, and thelike. See, for example, U.S. Pat. Nos. 5,350,674 and 5,585,362.

Chemical means for introducing a polynucleotide into a host cell includecolloidal dispersion systems, such as macromolecule complexes,nanocapsules, microspheres, beads, and lipid-based systems includingoil-in-water emulsions, micelles, mixed micelles, and liposomes. Apreferred colloidal system for use as a delivery vehicle in vitro and invivo is a liposome (i.e., an artificial membrane vesicle). Thepreparation and use of such systems is well known in the art. Exemplaryformulations are also disclosed in U.S. Pat. No. 5,981,505; U.S. Pat.Nos. 6,217,900; 6,383,512; U.S. Pat. No. 5,783,565; U.S. Pat. No.7,202,227; U.S. Pat. No. 6,379,965; U.S. Pat. No. 6,127,170; U.S. Pat.No. 5,837,533; and WO 03/093449, which are herein incorporated byreference in their entireties.

The present invention also provides compounds. These compoundsspecifically bind to at least one of the mutant ligand binding domainsof the α7 nAChR as described herein. In one embodiment, the compound hasthe structure of formula I:

or a pharmaceutically acceptable salt thereof, wherein A is one of:

wherein,each of R₁, R₄ and R₅ is independently selected from the group ofhydrogen, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ thioalkyl, C₁-C₆haloalkyl, C₁-C₆ thiohaloalkyl, C₁-C₆ haloalkoxy, amino, alkylamino,dialkylamino, alkylsulfonyl, aryl and heteroaryl, wherein said aryl andheteroaryl are optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆alkoxy, C₁-C₆ thioalkyl, C₁-C₆haloalkyl, C₁-C₆ thiohaloalkyl, C₁-C₆haloalkoxy, amino, alkylamino, dialkylamino or alkylsulfonyl;each of R₂ and R₃ is independently selected from the group of hydrogen,halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ thioalkyl, C₁-C₆ haloalkyl, C₁-C₆thiohaloalkyl, C₁-C₆ haloalkoxy, amino, alkylamino, dialkylamino,alkylsulfonyl, aryl and heteroaryl, wherein said aryl and heteroaryl areoptionally substituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆thioalkyl, C₁-C₆ haloalkyl, C₁-C₆ thiohaloalkyl, C₁-C₆haloalkoxy, amino,alkylamino, dialkylamino or alkylsulfonyl; orR₂ and R₃, together with the carbon atoms to which they are attached,form a 5 or 6-membered carbocyclic or heterocyclic ring optionallysubstituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ thioalkyl, C₁-C₆haloalkyl, C₁-C₆ thiohaloalkyl, C₁-C₆ haloalkoxy, amino, alkylamino,dialkylamino, alkylsulfonyl;with the provisos that: (a) at least one of R₁, R₂, R₃, R₄ and R₅ is nothydrogen; and (b) if neither of R₁ and R₅ is C₁-C₆ alkoxy, then R₃ ispresent and is not hydrogen.

In certain embodiments of the compound of formula I, A is

wherein R₃ is phenyl optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy or C₁-C₆ haloalkyl. In one such embodiment, each of R₁, R₂,R₄ and R₅ is hydrogen.

In certain other embodiments of the compound of formula I, A is

wherein R₃ is C₃-C₆ alkyl, C₅-C₆ alkoxy, C₁-C₂ haloalkoxy, C₁-C₂thiohaloalkyl, C₁-C₂ perhaloalkyl, dialkylamino or alkylsulfonyl. In onesuch embodiment, A is

wherein each of R₁, R₂, R₄ and R₅ is hydrogen. In another suchembodiment A is

wherein R₃ is C₁-C₂ perhaloalkyl or C₃-C₆ alkyl. In one such embodimenteach of R₄ and R₅ is hydrogen; R₁, if present, is hydrogen; and R₂, ifpresent, is hydrogen.

In one embodiment of the compound of formula I, at least one of R₁ andR₅ is methoxy, ethoxy or phenoxy. In one such embodiment, A is

wherein R₁ is methoxy, ethoxy or phenoxy; each of R₂, R₃ and R₄ isindependently hydrogen, chloro, fluoro, methoxy, ethoxy, methyl orethyl; and R₅ is hydrogen.

In another embodiment, the compound has the structure of formula II:

or a pharmaceutically acceptable salt thereof, wherein:A is aryl or heteroaryl optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy, C₁-C₆ thioalkyl, C₁-C₆ haloalkyl, furanyl or thiophenyl,wherein said furanyl and thiophenyl are optionally substituted withhalo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ thioalkyl or C₁-C₆ haloalkyl;each of R^(A) and R^(B) is hydrogen or C₁-C₆ alkyl;or R^(A) and R^(B) taken together are ═O; andn is 0 or 1.

In another embodiment, the compound has the structure of formula III:

whereineach

is a single or double bond;each R^(A) and R^(B) is hydrogen or R^(A) and R^(B) taken together are═O;

E is —N, —NH, CR^(C), or CR^(C)R^(D),

each R^(C) and R^(D) is hydrogen or R^(C) and R^(D) taken together are═O;or a pharmaceutically acceptable salt thereof;wherein said compound does not comprise adjacent double bonds.

In one such embodiment, the compound of formula III is a member selectedfrom the group of:

In one such embodiment, the compound of formula III is the compoundhaving the structure:

or a pharmaceutically acceptable salt thereof.

The compounds of formula I, II and III contain a chiral carbon atom, andmay be in the form of a mixture of the R enantiomer and the S enantiomer(e.g., a racemic mixture) or as substantially pure R or S enantiomer. Incertain embodiments of compounds of formula I, II and III, the Senantiomer is preferred. For example, in certain embodiments the Senantiomer exhibits greater selectivity than does the R enantiomer for aparticular mutated chimeric receptor of the invention than for thecorresponding wild type chimeric receptor.

As described above, the present invention provides a method of treatinga disease or disorder associated with the nervous system in a subject inneed thereof. In one embodiment, the method comprises delivering agenetic construct to a population of neurons in the subject, wherein thegenetic construct encodes a mutant α7 nicotinic acetylcholine receptor,wherein the mutation confers selective binding to a compound of formulaI, II or III as described above; and administering the compound to thesubject. The present invention also provides a method of modulating theexcitability of a neuronal cell. In one embodiment, the method comprisesexpressing in the neuronal cell a genetic construct encoding a chimericreceptor, wherein the chimeric receptor comprises a ligand bindingdomain from an α7 nicotinic acetylcholine receptor fused to atransmembrane domain from a ligand-gated ion channel protein, whereinsaid ligand binding domain comprises at least one mutation that confersselective binding to a compound of formula I, II or III as describedabove; and exposing the neuronal cell to the compound. In addition, thepresent invention provides kits comprising chimeric receptors of theinvention as disclosed herein and a compound of formula I, II or III asdescribed above.

In one such embodiment, the compound is represented by formula I,wherein A is

wherein R₃ is phenyl optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy or C₁-C₆ haloalkyl, each of R₁, R₂, R₄ and R₅ is hydrogen,and at least one mutation in the ligand binding domain of the mutant α7nicotinic acetylcholine receptor is W77F in SEQ ID NO: 1, SEQ ID NO: 6or SEQ ID NO: 10. For example, in one such embodiment, the mutatedchimeric receptor has the sequence of SEQ ID NO: 2, SEQ ID NO: 7 or SEQID NO: 11.

In another such embodiment, the compound is represented by formula I,wherein A is

wherein R₃ is C₃-C₆ alkyl, C₅-C₆ alkoxy, C₁-C₂ haloalkoxy, C₁-C₂thiohaloalkyl, C₁-C₂ perhaloalkyl, dialkylamino or alkylsulfonyl, eachof R₁, R₂, R₄ and R₅ is hydrogen, and at least one mutation in theligand binding domain of the mutant α7 nicotinic acetylcholine receptoris Q79G or Q79A in SEQ ID NO: 1, SEQ ID NO: 6 or SEQ ID NO: 10. Forexample, in one such embodiment, the mutated chimeric receptor has thesequence of SEQ ID NO: 3, SEQ ID NO: 8 or SEQ ID NO: 12.

In yet another such embodiment, the compound is represented by formulaI, wherein A is

wherein R₃ is C₁-C₂ perhaloalkyl or C₃-C₆ alkyl, each of R₄ and R₅ ishydrogen, R₁, if present, is hydrogen, R₂, if present, is hydrogen, andat least one mutation in the ligand binding domain of the mutant α7nicotinic acetylcholine receptor is Q79G or Q79A in SEQ ID NO: 1, SEQ IDNO: 6 or SEQ ID NO: 10. For example, in one such embodiment, the mutatedchimeric receptor has the sequence of SEQ ID NO: 3, SEQ ID NO: 8 or SEQID NO: 12.

In another such embodiment, the compound is represented by formula I,wherein A is one of:

wherein at least one of R₁ and R₅ is methoxy, ethoxy or phenoxy, and atleast one mutation in the ligand binding domain of the mutant α7nicotinic acetylcholine receptor is L141F or L141P in SEQ ID NO: 1, SEQID NO: 6 or SEQ ID NO: 10. For example, in one such embodiment, themutated chimeric receptor has the sequence of SEQ ID NO: 4, SEQ ID NO: 9or SEQ ID NO: 13.

In one such embodiment, A is

R₁ is methoxy, ethoxy or phenoxy, each of R₂, R₃ and R₄ is independentlyhydrogen, chloro, fluoro, methoxy, ethoxy, methyl or ethyl, and R₅ ishydrogen.

In another such embodiment, the compound is represented by formula II,wherein A is phenyl, pyridyl, pyrazinyl or quinoxalinyl, substitutedwith halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆thioalkyl or C₁-C₆ haloalkyl,and at least one mutation in the ligand binding domain of the mutant α7nicotinic acetylcholine receptor is L141F or L141P in SEQ ID NO: 1, SEQID NO: 6 or SEQ ID NO: 10. For example, in one such embodiment, themutated chimeric receptor has the sequence of SEQ ID NO: 4, SEQ ID NO: 9or SEQ ID NO: 13.

In yet another embodiment, the compound is represented by formula III,and at least one mutation in the ligand binding domain of the mutant α7nicotinic acetylcholine receptor is L141F or L141P in SEQ ID NO: 1, SEQID NO: 6 or SEQ ID NO: 10. For example, in one such embodiment, themutated chimeric receptor has the sequence of SEQ ID NO: 4, SEQ ID NO: 9or SEQ ID NO: 13. In one such embodiment, the compound is Compound No.212 as described herein.

“Alkyl” is hydrocarbon containing normal, secondary, tertiary or cycliccarbon atoms. For example, an alkyl group can have 1 to 20 carbon atoms(i.e, C₁-C₂₀ alkyl), 1 to 10 carbon atoms (i.e., C₁-C₁₀ alkyl), or 1 to6 carbon atoms (i.e., C₁-C₆ alkyl). Examples of suitable alkyl groupsinclude, but are not limited to, methyl (Me, —CH₃), ethyl (Et, —CH₂CH₃),1-propyl (n-Pr, n-propyl, —CH₂CH₂CH₃), 2-propyl (i-Pr, i-propyl,—CH(CH₃)₂), 1-butyl (n-Bu, n-butyl, —CH₂CH₂CH₂CH₃), 2-methyl-1-propyl(1-Bu, i-butyl, —CH₂CH(CH₃)₂), 2-butyl (s-Bu, s-butyl, —CH(CH₃)CH₂CH₃),2-methyl-2-propyl (t-Bu, t-butyl, —C(CH₃)₃), 1-pentyl (n-pentyl,—CH₂CH₂CH₂CH₂CH₃), 2-pentyl (—CH(CH₃)CH₂CH₂CH₃), 3-pentyl(—CH(CH₂CH₃)₂), 2-methyl-2-butyl (—C(CH₃)₂CH₂CH₃), 3-methyl-2-butyl(—CH(CH₃)CH(CH₃)₂), 3-methyl-1-butyl (—CH₂CH₂CH(CH₃)₂), 2-methyl-1-butyl(—CH₂CH(CH₃)CH₂CH₃), 1-hexyl (—CH₂CH₂CH₂CH₂CH₂CH₃), 2-hexyl(—CH(CH₃)CH₂CH₂CH₂CH₃), 3-hexyl (—CH(CH₂CH₃)(CH₂CH₂CH₃)),2-methyl-2-pentyl (—C(CH₃)₂CH₂CH₂CH₃), 3-methyl-2-pentyl(—CH(CH₃)CH(CH₃)CH₂CH₃), 4-methyl-2-pentyl (—CH(CH₃)CH₂CH(CH₃)₂),3-methyl-3-pentyl (—C(CH₃)(CH₂CH₃)₂), 2-methyl-3-pentyl(—CH(CH₂CH₃)CH(CH₃)₂), 2,3-dimethyl-2-butyl (—C(CH₃)₂CH(CH₃)₂),3,3-dimethyl-2-butyl (—CH(CH₃)C(CH₃)₃, and octyl (—(CH₂)₇CH₃).

“Alkoxy” means a group having the formula —O-alkyl, in which an alkylgroup, as defined above, is attached to the parent molecule via anoxygen atom. The alkyl portion of an alkoxy group can have 1 to 20carbon atoms (i.e., C₁-C₂₀ alkoxy), 1 to 12 carbon atoms (i.e., C₁-C₁₂alkoxy), or 1 to 6 carbon atoms (i.e., C₁-C₆ alkoxy). Examples ofsuitable alkoxy groups include, but are not limited to, methoxy (—O—CH₃or OMe), ethoxy (—OCH₂CH₃ or —OEt), t-butoxy (—O—C(CH₃)₃ or —OtBu) andthe like.

“Aryl” means a monovalent aromatic hydrocarbon radical derived by theremoval of one hydrogen atom from a single carbon atom of a parentaromatic ring system. For example, an aryl group can have 6 to 20 carbonatoms, 6 to 14 carbon atoms, or 6 to 12 carbon atoms. Typical arylgroups include, but are not limited to, radicals derived from benzene(e.g., phenyl), substituted benzene, naphthalene, anthracene, biphenyl,and the like. “Aryloxy” means a group having the formula —O-aryl, inwhich an aryl group, as defined above, is attached to the parentmolecule via an oxygen atom.

“Heteroalkyl” refers to an alkyl group where one or more carbon atomshave been replaced with a heteroatom, such as, O, N, or S. For example,if the carbon atom of the alkyl group which is attached to the parentmolecule is replaced with a heteroatom (e.g., O, N, or S) the resultingheteroalkyl groups are, respectively, an alkoxy group (e.g., —OCH₃,etc.), an amine (e.g., —NHCH₃, —N(CH₃)₂, etc.), or a thioalkyl group(e.g., —SCH₃). If a non-terminal carbon atom of the alkyl group which isnot attached to the parent molecule is replaced with a heteroatom (e.g.,O, N, or S) and the resulting heteroalkyl groups are, respectively, analkyl ether (e.g., —CH₂CH₂—O—CH₃, etc.), an alkyl amine (e.g.,—CH₂NHCH₃, —CH₂N(CH₃)₂, etc.), or a thioalkyl ether (e.g., —CH₂—S—CH₃).If a terminal carbon atom of the alkyl group is replaced with aheteroatom (e.g., O, N, or S), the resulting heteroalkyl groups are,respectively, a hydroxyalkyl group (e.g., —CH₂CH₂—OH), an aminoalkylgroup (e.g., —CH₂NH₂), or an alkyl thiol group (e.g., —CH₂CH₂—SH). Aheteroalkyl group can have, for example, 1 to 20 carbon atoms, 1 to 10carbon atoms, or 1 to 6 carbon atoms. A C₁-C₆ heteroalkyl group means aheteroalkyl group having 1 to 6 carbon atoms. “Heteroalkoxy” means agroup having the formula —O-heteroalkyl, in which a heteroalkyl group,as defined above, is attached to the parent molecule via an oxygen atom.

“Heterocycle” or “heterocyclyl” as used herein includes by way ofexample and not limitation those heterocycles described in Paquette, LeoA.; Principles of Modern Heterocyclic Chemistry (W. A. Benjamin, NewYork, 1968), particularly Chapters 1, 3, 4, 6, 7, and 9; The Chemistryof Heterocyclic Compounds, A Series of Monographs” (John Wiley & Sons,New York, 1950 to present), in particular Volumes 13, 14, 16, 19, and28; and J. Am. Chem. Soc. (1960) 82:5566. In one specific embodiment ofthe invention “heterocycle” includes a “carbocycle” as defined herein,wherein one or more (e.g. 1, 2, 3, or 4) carbon atoms have been replacedwith a heteroatom (e.g. O, N, or S). The terms “heterocycle” or“heterocyclyl” includes saturated rings, partially unsaturated rings,and aromatic rings (i.e., heteroaromatic rings). Substitutedheterocyclyls include, for example, heterocyclic rings substituted withany of the substituents disclosed herein including carbonyl groups. Anon-limiting example of a carbonyl substituted heterocyclyl is:

Examples of heterocycles include by way of example and not limitationpyridyl, dihydroypyridyl, tetrahydropyridyl (piperidyl), thiazolyl,tetrahydrothiophenyl, sulfur oxidized tetrahydrothiophenyl, pyrimidinyl,furanyl, thienyl, pyrrolyl, pyrazolyl, imidazolyl, tetrazolyl,benzofuranyl, thianaphthalenyl, indolyl, indolenyl, quinolinyl,isoquinolinyl, benzimidazolyl, piperidinyl, 4-piperidonyl, pyrrolidinyl,2-pyrrolidonyl, pyrrolinyl, tetrahydrofuranyl, tetrahydroquinolinyl,tetrahydroisoquinolinyl, decahydroquinolinyl, octahydroisoquinolinyl,azocinyl, triazinyl, 6H-1,2,5-thiadiazinyl, 2H,6H-1,5,2-dithiazinyl,thienyl, thianthrenyl, pyranyl, isobenzofuranyl, chromenyl, xanthenyl,phenoxathinyl, 2H-pyrrolyl, isothiazolyl, isoxazolyl, pyrazinyl,pyridazinyl, indolizinyl, isoindolyl, 3H-indolyl, 1H-indazoly, purinyl,4H-quinolizinyl, phthalazinyl, naphthyridinyl, quinoxalinyl,quinazolinyl, cinnolinyl, pteridinyl, 4aH-carbazolyl, carbazolyl,β-carbolinyl, phenanthridinyl, acridinyl, pyrimidinyl, phenanthrolinyl,phenazinyl, phenothiazinyl, furazanyl, phenoxazinyl, isochromanyl,chromanyl, imidazolidinyl, imidazolinyl, pyrazolidinyl, pyrazolinyl,piperazinyl, indolinyl, isoindolinyl, quinuclidinyl, morpholinyl,oxazolidinyl, benzotriazolyl, benzisoxazolyl, oxindolyl, benzoxazolinyl,isatinoyl, and bis-tetrahydrofuranyl:

By way of example and not limitation, carbon bonded heterocycles arebonded at position 2, 3, 4, 5, or 6 of a pyridine, position 3, 4, 5, or6 of a pyridazine, position 2, 4, 5, or 6 of a pyrimidine, position 2,3, 5, or 6 of a pyrazine, position 2, 3, 4, or 5 of a furan,tetrahydrofuran, thiofuran, thiophene, pyrrole or tetrahydropyrrole,position 2, 4, or 5 of an oxazole, imidazole or thiazole, position 3, 4,or 5 of an isoxazole, pyrazole, or isothiazole, position 2 or 3 of anaziridine, position 2, 3, or 4 of an azetidine, position 2, 3, 4, 5, 6,7, or 8 of a quinoline or position 1, 3, 4, 5, 6, 7, or 8 of anisoquinoline. Still more typically, carbon bonded heterocycles include2-pyridyl, 3-pyridyl, 4-pyridyl, 5-pyridyl, 6-pyridyl, 3-pyridazinyl,4-pyridazinyl, 5-pyridazinyl, 6-pyridazinyl, 2-pyrimidinyl,4-pyrimidinyl, 5-pyrimidinyl, 6-pyrimidinyl, 2-pyrazinyl, 3-pyrazinyl,5-pyrazinyl, 6-pyrazinyl, 2-thiazolyl, 4-thiazolyl, or 5-thiazolyl.

By way of example and not limitation, nitrogen bonded heterocycles arebonded at position 1 of an aziridine, azetidine, pyrrole, pyrrolidine,2-pyrroline, 3-pyrroline, imidazole, imidazolidine, 2-imidazoline,3-imidazoline, pyrazole, pyrazoline, 2-pyrazoline, 3-pyrazoline,piperidine, piperazine, indole, indoline, 1H-indazole, position 2 of aisoindole, or isoindoline, position 4 of a morpholine, and position 9 ofa carbazole, or β-carboline. Still more typically, nitrogen bondedheterocycles include 1-aziridyl, 1-azetedyl, 1-pyrrolyl, 1-imidazolyl,1-pyrazolyl, and 1-piperidinyl.

“Heteroaryl” refers to a monovalent aromatic heterocyclyl having atleast one heteroatom in the ring. Non-limiting examples of suitableheteroatoms which can be included in the aromatic ring include oxygen,sulfur, and nitrogen. Non-limiting examples of heteroaryl rings includeall of those listed in the definition of “heterocyclyl”, includingpyridinyl, pyrrolyl, oxazolyl, indolyl, isoindolyl, purinyl, furanyl,thienyl, benzofuranyl, benzothiophenyl, carbazolyl, imidazolyl,thiazolyl, isoxazolyl, pyrazolyl, isothiazolyl, quinolyl, isoquinolyl,pyridazyl, pyrimidyl, pyrazyl, etc. “Heteroaryloxy” means a group havingthe formula —O-heteroaryl, in which a heteroaryl group, as definedabove, is attached to the parent molecule via an oxygen atom.

An “acyl group” or “alkanoyl” is a functional group having the basicformula RC(═O) with a double bond between the carbon and oxygen atoms(i.e. a carbonyl group), and a single bond between R and the carbon. An“O-acyl group” is an acyl group as defined above linked to the parentmolecule via an oxygen atom.

A “halogen” or halide is a fluorine, chlorine, bromine, iodine, orastatine atom.

In some embodiments, the compound has the structure in formula I,wherein R₂ is a hydrogen and R₄ is a hydrogen. In other embodiments, thecompound has the structure in formula I, wherein R₁ is a methoxy,ethoxy, or phenoxy group, R₂ is a hydrogen and R₄ is a hydrogen. Instill other embodiments, the compound has the structure in formula I,wherein R₁ is a methoxy, ethoxy, or phenoxy group, R₂ is a hydrogen, R₃is a hydrogen, methyl, methoxy, or chloride group, and R₄ is a hydrogen.In one embodiment, the compound has the structure in formula I, whereinR₁ is a methoxy group, R₂ is a hydrogen, R₃ is a hydrogen, and R₄ is amethoxy group. In certain embodiments, the compound is 85S, 86S, 87S,88S, 89S, 90S or 91S.

The present invention encompasses the use of pharmaceutical compositionsof the appropriate vector encoding the chimeric receptors as well aspharmaceutical compositions of the synthetic ligands to practice themethods of the invention. Where clinical applications are contemplated,pharmaceutical compositions will be prepared in a form appropriate forthe intended application. Generally, this will entail preparingcompositions that are essentially free of pyrogens, as well as otherimpurities that could be harmful to humans or animals.

Compositions of the present invention comprise an effective amount ofactive ingredient (the genetic construct or synthetic ligand), dissolvedor dispersed in a pharmaceutically acceptable carrier or aqueous medium.The phrases “pharmaceutically acceptable” or “pharmacologicallyacceptable” refer to molecular entities and compositions that do notproduce adverse, allergic, or other untoward reactions when administeredto an animal or a human. As used herein, “pharmaceutically acceptablecarrier” includes solvents, buffers, solutions, dispersion media,coatings, antibacterial and antifungal agents, isotonic and absorptiondelaying agents and the like acceptable for use in formulatingpharmaceuticals, such as pharmaceuticals suitable for administration tohumans. The use of such media and agents for pharmaceutically activesubstances is well known in the art. Except insofar as any conventionalmedia or agent is incompatible with the active ingredients of thepresent invention, its use in therapeutic compositions is contemplated.Supplementary active ingredients also can be incorporated into thecompositions, provided they do not inactivate the active ingredient(genetic constructs or synthetic ligands) of the compositions.

Some examples of materials which can serve as pharmaceuticallyacceptable carriers include, but are not limited to, sugars such aslactose, glucose and sucrose; starches such as corn starch and potatostarch; cellulose and its derivatives such as sodium carboxymethylcellulose, ethyl cellulose and cellulose acetate; powdered tragacanth;malt; gelatin; talc; excipients such as cocoa butter and suppositorywaxes; oils such as peanut oil, cottonseed oil; safflower oil; sesameoil; olive oil; corn oil and soybean oil; glycols; such a propyleneglycol; esters such as ethyl oleate and ethyl laurate; agar; bufferingagents such as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol,and phosphate buffer solutions, as well as other non-toxic compatiblelubricants such as sodium lauryl sulfate and magnesium stearate, as wellas coloring agents, releasing agents, coating agents, sweetening,flavoring and perfuming agents, preservatives and antioxidants can alsobe present in the composition, according to the judgment of theformulator.

The active compositions of the present invention may include classicpharmaceutical preparations. Administration of these compositionsaccording to the present invention may be via any common route so longas the target tissue (e.g., neuronal population) is available via thatroute. This includes oral, nasal, or buccal. Alternatively,administration may be by intradermal, subcutaneous, intramuscular,intrathecal, intracerebral, intraperitoneal or intravenous injection.Such compositions would normally be administered as pharmaceuticallyacceptable compositions, as described above.

By way of illustration, solutions of the active compounds as free baseor pharmacologically acceptable salts can be prepared in water suitablymixed with a surfactant, such as hydroxypropylcellulose. Dispersions canalso be prepared in glycerol, liquid polyethylene glycols, and mixturesthereof and in oils. Under ordinary conditions of storage and use, thesepreparations generally contain a preservative to prevent the growth ofmicroorganisms.

The pharmaceutical forms suitable for injectable use include, forexample, sterile aqueous solutions or dispersions and sterile powdersfor the extemporaneous preparation of sterile injectable solutions ordispersions. Generally, these preparations are sterile and fluid to theextent that easy injectability exists. Preparations should be stableunder the conditions of manufacture and storage and should be preservedagainst the contaminating action of microorganisms, such as bacteria andfungi. Appropriate solvents or dispersion media may contain, forexample, water, ethanol, polyol (for example, glycerol, propyleneglycol, and liquid polyethylene glycol, and the like), suitable mixturesthereof, and vegetable oils. The proper fluidity can be maintained, forexample, by the use of a coating, such as lecithin, by the maintenanceof the required particle size in the case of dispersion and by the useof surfactants. The prevention of the action of microorganisms can bebrought about by various antibacterial and antifungal agents, forexample, parabens, chlorobutanol, phenol, sorbic acid, thimerosal, andthe like. In many cases, it will be preferable to include isotonicagents, for example, sugars or sodium chloride. Prolonged absorption ofthe injectable compositions can be brought about by the use in thecompositions of agents delaying absorption, for example, aluminummonostearate and gelatin.

Sterile injectable solutions may be prepared by incorporating the activecompounds in an appropriate amount into a solvent along with any otheringredients (for example as enumerated above) as desired, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the various sterilized active ingredients into a sterilevehicle which contains the basic dispersion medium and the desired otheringredients, e.g., as enumerated above. In the case of sterile powdersfor the preparation of sterile injectable solutions, the preferredmethods of preparation include vacuum-drying and freeze-dryingtechniques which yield a powder of the active ingredient(s) plus anyadditional desired ingredient from a previously sterile-filteredsolution thereof.

The compositions of the present invention generally may be formulated ina neutral or salt form. The salts can be prepared in situ during thefinal isolation and purification of the compounds of the invention, orseparately by reacting the free base function with a suitable organicacid. Examples of pharmaceutically acceptable, nontoxic acid additionsalts are salts of an amino group formed with inorganic acids such ashydrochloric acid, hydrobromic acid, phosphoric acid, sulfuric acid andperchloric acid or with organic acids such as acetic acid, oxalic acid,maleic acid, tartaric acid, citric acid, succinic acid or malonic acidor by using other methods used in the art such as ion exchange. Otherpharmaceutically acceptable salts include adipate, alginate, ascorbate,aspartate, benzenesulfonate, benzoate, bisulfate, borate, butyrate,camphorate, camphorsulfonate, citrate, cyclopentanepropionate,digluconate, dodecylsulfate, ethanesulfonate, formate, fumarate,glucoheptonate, glycerophosphate, gluconate, hernisulfate, heptanoate,hexanoate, hydroiodide, 2-hydroxy-ethanesulfonate, lactobionate,lactate, laurate, lauryl sulfate, malate, maleate, malonate,methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, oleate,oxalate, palmitate, pamoate, pectinate, persulfate, 3-phenylpropionate,phosphate, picrate, pivalate, propionate, stearate, succinate, sulfate,tartrate, thiocyanate, p-toluenesulfonate, undecanoate, valerate salts,and the like. Representative alkali or alkaline earth metal saltsinclude sodium, lithium, potassium, calcium, magnesium, and the like.Further pharmaceutically acceptable salts include, when appropriate,nontoxic ammonium, quaternary ammonium, and amine cations formed usingcounterions such as halide, hydroxide, carboxylate, sulfate, phosphate,nitrate, loweralkyl sulfonate and aryl sulfonate. Salts formed with freecarboxyl groups of the compound can also be derived from inorganic bases(e.g., sodium, potassium, ammonium, calcium, or ferric hydroxides) orfrom organic bases (e.g., isopropylamine, trimethylamine, histidine,procaine and the like.

Upon formulation, solutions are preferably administered in a mannercompatible with the dosage formulation and in such amount as istherapeutically effective. The formulations may easily be administeredin a variety of dosage forms such as injectable solutions, drug releasecapsules and the like. For parenteral administration in an aqueoussolution, for example, the solution generally is suitably buffered andthe liquid diluent first rendered isotonic for example with sufficientsaline or glucose. Such aqueous solutions may be used, for example, forintravenous, intramuscular, subcutaneous, intrathecal, intracerebral,and intraperitoneal administration. Preferably, sterile aqueous mediaare employed as is known to those of skill in the art, particularly inlight of the present disclosure. By way of illustration, a single dosemay be dissolved in 1 ml of isotonic NaCl solution and either added to1000 ml of hypodermoclysis fluid or injected at the proposed site ofinfusion, (see for example, “Remington's Pharmaceutical Sciences” 15thEdition, pages 1035-1038 and 1570-1580). Some variation in dosage willnecessarily occur depending on the condition of the subject beingtreated and the particular active ingredient (e.g., synthetic ligand orgenetic construct) used. The person responsible for administration will,in any event, determine the appropriate dose for the individual subject.

The present invention also encompasses a kit comprising a chimericreceptor of the invention as disclosed herein and at least one compoundligand. The kit may include a genetic construct that encodes thechimeric receptor and optionally instructions for expressing the geneticconstruct in a host cell or tissue.

In one embodiment, the kit contains a chimeric receptor and at least onecompound, wherein the ligand binding domain of the chimeric receptor hasa W77F mutation in SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 10 thatconfers selective binding to the compound. In some embodiments, thecompound is 28S, 34S, 96R, 97R, 131S, 132S, 278S, 279S, and/or 281S. Inother embodiments, the compound is 28S, 34S and/or 132S. In anotherembodiment, the kit contains a chimeric receptor and a compound, whereinthe ligand binding domain of the chimeric receptor has a Q79A or Q79Gmutation in SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 10 that confersselective binding to the compound. In certain embodiments, the compoundis 9S, 16S, 22S, 38R, 115S, 117S, 134S, 148S, 149S, 154S, 156S, 157S,158S, 163S, 164S, 165S, 170R, 292R, 295S, and/or 296S. In otherembodiments, the compound is 9S, 16S, 22S, 38R, and/or 165S. In stillanother embodiment, the kit contains a chimeric receptor and a compound,wherein the ligand binding domain of the chimeric receptor has a L141For L141P mutation in SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO: 10 thatconfers selective binding to the compound. In some embodiments, thecompound is 19S, 85S, 86S, 88S, 89S, 90S, 91S, 1185, 119S, 120S, 121S,127S, 208S, 212, 241, 242, 245, 253, 254, 255, and/or 294S. In otherembodiments, the compound is 19S, 85S, 86S, 88S, 89S, 90S, and/or 118S.

The present invention also includes a method of treating a disease ordisorder associated with the nervous system in a subject in needthereof. In one embodiment, the method comprises delivering a geneticconstruct to a population of neurons in the subject, wherein the geneticconstruct encodes a chimeric receptor, wherein the chimeric receptorcomprises a ligand binding domain from an α7 nicotinic acetylcholinereceptor fused to a transmembrane domain from a ligand-gated ion channelprotein, wherein said ligand binding domain comprises at least onemutation that confers selective binding to a compound; and administeringthe compound to the subject. Preferably, the compound is capable ofpassing through the blood brain barrier.

Such therapeutic methods find use in disease states or otherphysiological conditions in which the disease or symptoms can beimproved by alteration in neuronal activity. In conditions in whichdecreasing activity of a subpopulation of neurons provides a therapeuticbenefit, an anion-conducting chimeric receptor of the invention (e.g.,α7-GlyR or α7-GABA C) may be expressed in such neurons and subsequentlyactivated with its tailored compound to silence these neurons. In suchembodiments, the activity of the population of neurons is decreasedfollowing administration of the compound to the subject. Conversely, inconditions in which increasing activity of a neuronal subpopulationprovides a therapeutic benefit, a cationic-selective chimeric receptorof the invention (e.g., α7-5HT3) may be expressed in such neurons andsubsequently activated with its tailored compound to increase actionpotential frequency in the neurons. In these embodiments, the activityof the population of neurons is increased following administration ofthe compound to the subject.

By way of example, chronic pain can be treated in a subject with themethods of the invention. Painful stimuli are primarily sensed andpropagated to the nervous system by neurons that give rise to axons anddendrites that are referred to as C-fibers. The cell bodies of theseneurons are in the dorsal root ganglia, peripheral to the spinal cord.These cell bodies express genes, Substance P and TRPV1, selectively overother neurons in the dorsal root ganglia which pass sensory informationsuch as touch. By using a gene delivery technique as described herein(e.g., a viral vector) to target DNA encoding neuronal silencers suchas, but not limited to, α7-GlyR L141F or α7-GABA C L141F under controlof either of the substance P or TRPV1 promoters, selective expression ofthe ion channels is achieved. Once expressing these ion channels, theactivity of these neurons can be selectively reduced in the presence ofa ligand for these chimeric receptors, such as 89S. Silencing C-fibersblocks painful stimuli from being detected. This approach permits theC-fibers to be selectively silenced without silencing the associated Aand delta fibers which are associated with sensing touch. Silencing theC-fibers using less specific techniques than those described here wouldresult in undesired loss of sensation such as numbness instead ofselective block of pain.

The invention could also be applied to treatment of epilepsy, which is adisorder of excessive neuronal activity in the brain ultimately leadingto seizures. In epilepsy, seizures can result from neuronal overactivityin a specific brain region or foci that can then spread to other brainregions. Neuronal silencers, such as α7-GlyR L141F or α7-GABA C L141F,can be specifically targeted to these overactive brain regions (e.g.,neurons in the seizure focus) using viral vectors or other geneticconstructs encoding the chimeric channels under the control ofneuron-specific promoter fragments, such as the synapsin promoter orneuron-specific enolase promoter. Expression of these channels in theregion of interest renders these neurons sensitive to systemicallyadministered synthetic ligands of the invention, such as compound 89,which would silence this brain region only when required (e.g., if aseizure was developing). This would leave the rest of the brainunperturbed, significantly reducing side effects associated withepilepsy therapy. The above-described examples were to illustrateparticular embodiments and not to limit the invention in any way.Similar methods could be used to treat other disorders or diseasesassociated with the nervous system including, but not limited to,Alzheimer's Disease, Parkinson's Disease, Schizophrenia, hypothalamicdisorders, and Huntington's Disease.

The examples which follow are set forth to illustrate the presentinvention, and are not to be construed as limiting thereof.

EXAMPLES Example 1 Generation of Mutants of the α7 NicotinicAcetylcholine Receptor Ligand Binding Domain

To engineer non-natural pharmacology into the ligand binding domain(LBD) of the α7 nicotinic acetylcholine receptor (nAChR), a model of theligand-ion channel interaction was developed. This homology model wasbased on the x-ray crystal structure of the snail acetylcholine bindingprotein (AChBP) bound to nicotine (Celie et al. (2004) Neuron, Vol. 41:907-914). The homology model was generated by aligning the sequence fromthe rat α7 nAChR with the crystal structure of Lymnaea stagnalis (greatpond snail) AchBP bound to nicotine (PDB 1UW6) using PROMALS3D(http://prodata.swmed.edu/promals3d). All computational modeling wasdone within the Chameleon software package. Insertions and deletions inthe α7 nAChR sequence relative to AchBP were modeled by search of theProtein Data Bank (PDB) for secondary structure elements with similartermini, using a sliding window through the α7 sequence. Proteinside-chains were remodeled using a backbone-independent rotamer libraryas previously described (Lovell et al. (2000) “The Penultimate RotamerLibrary”, Proteins: Structure Function and Genetics, Vol. 40: 389-408;Lovell et al. (2003) “Structure Validation by Cα Geometry: φ,ψ and CβDeviation”, Proteins: Structure, Function and Genetics, Vol. 50:437-450), with manually-added diversity.

PNU-282987, a selective α7 nAChR agonist, was docked into this homologymodel, with a binding mode analogous to nicotine, where the protonatedtertiary amine interacts with W171 (See FIG. 1C). Note that the aminoacid numbering is based on translation of the cDNA sequence used hereand includes the signal peptide. The PNU molecule was modeled usingChem3D (CambridgeSoft) and semi-manually docked into the α7 nAChRbinding pocket. Specifically, the quaternary amine was superposed ontothat of the nicotine molecule, and allowed to rotate freely around theH-N axis, thus preserving this “pharmacophoric” hydrogen bond with thebackbone carbonyl of AchBP/α7 nAChR. A minimum-energy conformation wasdetermined, and then the surrounding protein side-chains were placedagain in the context of the PNU molecule. The resulting model of thecomplex was screened for clashes using MolProbity(http://molprobity.biochem.duke.edu/), which were fixed by iterativeapplication of main-chain “backrub” and side-chain optimization. PNUderivatives were also modeled in Chem3D; mutant structures were obtainedby selective replacement of single side-chains. All images were producedusing PyMOL (http://pymol.sourceforge.net/).

Based on previously reported structure activity relationships ofbenzamide quinuclidines with the α7-5HT3a chimeric receptor (Bodnar, etal. (2005) Journal of Medicinal Chemistry, Vol. 48: 905-908), weconcluded that bulky substitution of the benzamide was detrimental toreceptor activity. Therefore, to design α7 nAChR LBDs with selectivityfor small molecule agonists, LBD mutations that would accommodate thesebulky groups were identified. Mutations were made in amino acids thatwere adjacent to the benzamide functionality in the homology model (e.g.W77, Q79, Q139, L141; see FIG. 1C). Although the model called forreducing steric bulk in the LBD in order to accommodate bulkier ligands,the modifications of these residues were not limited solely to smalleramino acids. Instead, each of these positions were mutated to one of 19alternative amino acids generating a total of 76 single mutant channels.See X axis of FIG. 1G for a list of all 76 mutations. This mutationalapproach was adopted to account for complex interactions in proteinstructure that can lead to unexpected conformational effects, which mayalso favor binding of a larger ligand.

Site-directed mutagenesis of a chimeric receptor containing the ligandbinding domain from the human α7 nAChR fused to the transmembrane domainof the mouse serotonergic ionotropic receptor (5HT3) was performed usingstandard techniques to produce each of the 76 single channel mutantsdescribed above. The wild-type amino acid sequence for the human/mouseα7-5HT3 chimeric receptor is shown in FIG. 10 (SEQ ID NO: 1).

Example 2 Functional Characterization of α7 nAChR-5HT3 Chimeric ReceptorMutants

As described in Example 1, the human/mouse α7-5HT3 chimeric receptor wasused as a template to generate the point mutations in the α7 LBD. Thischimeric receptor expresses well in heterologous cell culture systemsand it exhibits slower desensitization kinetics relative to the nativeα7 nAChR (Eisele et al. (1993) Nature, Vol. 366: 479-483). HEK 293 cells(ATCC CRL-1573) were transfected with a single plasmid encoding one ofthe seventy six single α7-5HT3 receptor mutants using a commercialtransfection reagent (FuGene HD, Roche Applied Science). Each of theseventy six different α7-5HT3 receptor mutants were prescreened for ionchannel function and cell surface expression.

Ion channel function was measured using a commercially availablefluorescence-based membrane potential (MP) assay (MDS AnalyticalTechnologies). Briefly, the MP assay involves loading cells expressingthe chimeric receptors with a voltage-sensitive dye, which fluoresces ata particular wavelength upon changes in the cells' membrane potential.Increases in fluorescence correlate with ion flux into the cell, andthus provide a measure of ion channel activity. HEK cells expressingeither the wild type α7-5HT3 chimeric receptor or one of the singlemutant receptors were exposed to a single concentration of threedifferent conventional ligands for the α7 nAChR: acetylcholine (Ach),nicotine, and PNU-282987 (FIG. 1B). Whole cell voltage clamp recordingsof α7-5HT3 responses to PNU-282987 show an initial peak current(I_(peak)) which desensitizes (t_(1/2)=0.26±0.05 sec) to a steady statecurrent (I_(SS)) (FIG. 1D). Because the ultimate application of thischannel as a tool with ligand application in times of minutes, I_(SS) isthe most relevant value for assessing ligand efficacy. For highthroughput measurements of ion channel function, we used a platereader-compatible fluorescence-based membrane potential (MP) assay (FIG.1E). The dose response curves from this assay reflected I_(SS) resultingfrom sustained ligand activation of the channel (FIG. 1F). EC50s forI_(peak) measured in whole cell voltage clamp were typically 3-10 foldhigher than for I_(SS) (FIG. 1F, Table 2). Thus, the MP assay is asuitable method for measuring ion channel activation of the α7-5HT3chimeric receptors. For each of the seventy six mutant chimericreceptors, responses to ACh (100 μM), nicotine (100 μM), and PNU-282987(10 μM) measured in the MP assay were normalized to the response to ACh(100 μM). The results are shown in the colormap in FIG. 1G.

Cell surface expression of the wild-type and mutant α7-5HT3 chimericreceptors was determined using a fluorescence assay for surface-boundAlexa-594 labeled α-bungarotoxin. The relative amount of cell surfaceα-bungarotoxin binding sites (e.g. cell surface expression of thechimeric receptors) was measured by normalizing the fluorescentintensity of Alexa-594 labeled α-bungarotoxin bound to cells expressingα7-5HT3 mutant chimeric receptors relative to cells expressing α7-5HT3under non-permeablizing conditions. Mutant chimeric receptorsdemonstrating >40% surface expression and >50% activity with any of thethree ligands were used for additional dose response assays (FIG. 1G).

Example 3 Identification of Selective Ligands for Mutant α7 nAChR-5HT3Chimeric Receptors

A focused 71-membered library of 3-aminoquinuclidine benzamides wassynthesized (FIG. 2). Representative chemical synthesis procedures forsome of the compounds in the library are listed at the end of thisExample. It was previously reported that α7-5HT3 is preferentiallyactivated by the R enanantiomer of 3-aminoquinuclidine benzamide (Bodnaret al. (2005) Journal of Medicinal Chemistry, Vol. 48: 905-908).Furthermore, the 2′-substitution of the benzamide and bulky 4′-benzamidesubstituents were reported to most adversely affect activity of theα7-5HT3 chimeric receptor. Thus, we biased our library of moleculestowards substitutents that would be least active against the α7-5HT3“wild type” (wt) receptor. Additionally, the S enantiomers for many ofthe compounds were also synthesized.

Using the MP assay, we measured 1118 dose response curves against 43α7-5HT3 single amino acid mutations and the 3-aminoquinuclidinebenzamides as well as ACh, nicotine, and PNU-282987 (FIG. 3). Notably,several mutant ion channel-ligand combinations emerged showing selectiveactivity for the mutant chimeric receptor as compared to the wild-typeα7-5HT3 receptor (FIG. 4). In most cases, the S enantiomer of aparticular compound conferred the greatest selectivity over thewild-type α7-5HT3 chimeric receptor. One exception was the interactionof 38R with α7-5HT3 Q79G (EC50 =1.6 μM, no activity against α7-5HT3 wt).We selected W77F (SEQ ID NO: 2), Q79G (SEQ ID NO: 3), and L141F (SEQ IDNO: 4) mutant α7-5HT3 chimeric receptors for further investigation. Thedose response curves revealed a specific interaction of the W77F, Q79Gand L141F mutant chimeric receptors with compounds 28S, 34S, 9S, 22S,38R, and 19S (FIG. 4). EC50s for activating these mutant chimericreceptors for these three compounds were in the range of 0.8-3 μM.Importantly, these compounds (28S, 34S, 9S, 22S, 38R, and 19S) did notinduce activate the unmodified α7-5HT3 receptor. In addition to thesecompounds, there were a number of selective interactions of α7-5HT3mutant receptors with compounds from the library exhibiting EC50s around10 μM.

Several ligand-ion channel combinations also showed selectivity relativeto each another (FIG. 5). Compound 132S (EC50_(MP)=4.3±0.5 μM), afluorinated analog of 28S, selectively activated α7-5HT3 W77F overreceptors with the Q79G and L141F mutations. Similarly, 22S and 89S wereselective for Q79G and L141F, respectively. Such selectivity is acritical feature for developing tools that allows separate manipulationof multiple neuron populations in the same organism.

To improve the selective interaction of 19S with the α7-5HT3 L141Fmutant chimeric receptor, we synthesized and tested eight3-aminoquinuclidine-2′-alkoxybenzamide analogs (Table 1). The dimethoxyderivatives, 88S and 89S, exhibited 5-10 fold improvement in efficacyagainst this mutant receptor (EC50_(MP), 88S: 0.1±0.1 μM; 89S: 0.4±0.1μM) while exhibiting no activity against the α7-5HT3 wt receptor.

TABLE 1 Structure activity relationships of analogs of compound 19Sagainst α7-5HT3 L141F

Ligand-receptor affinities could also be improved with additionalmutations in the ligand binding site. Analysis of dose response curvesfrom α7-5HT3 Q139M channels showed higher efficacy for a number ofligands that activated α7-5HT3 wt. However, there is little selectivityconferred by this mutation. We reasoned that in combination with theselectivity mutations described above, the identified interactions couldbe improved. In some cases these binding site mutations were compatible,and α7-5HT3 Q79G Q139M showed, relative to α7-5HT3 Q79G, 8- and 20-foldimprovement in efficacy over with 38R (EC50_(MP)=0.23±0.07 μM) and 22S(EC50_(MP)=0.06±0.04 μM), respectively (Table 2, FIG. 6).

Example 4 Efficacy of Mutant α7 nAChR and Chimeric Receptors by CompoundLigands

Several series of small molecules were prepared and the activity andselectivity of the molecules were compared against binding to wild typeα7-5HT3. Compounds 28S, 34S, 96R, 97R, 131S, 132S, 278S, 279S, and 281Seach have EC50 values of about 100 μM for wt α7-5HT3 and of betweenabout 0.8 and about 7.7 μM for α7-5HT3 W77F. Compounds 165S, 165, 115S,163S, 117S, 154S, 149S, 164S, 22S, 157S, 170R, 156S, 295S, 292R, 296S,38R, 134S, and 148S each have EC50 values of about 100 μM for wt α7-5HT3and EC50 values of between about 0.8 and about 7.8 μM for α7-5HT3 Q79G.Compounds 19S, 85S, 86S, 88S, 89S, 90S, 91S, 118S, 119S, 120S, 121S,127S, and 294S each have EC50 values of about 100 μM for wt α7-5HT3 andEC50 values of between about 0.1 and about 3.4 μM for α7-5HT3 L141F.Compounds 212, 241, 242, 253, 254, and 255 each have EC50 values ofabout 100 μM for wt α7-5HT3 and EC50 values of between about 0.1 andabout 15 μM for α7-5HT3 L141F.

Table 2 shows representative EC50 values of various synthetic ligandsfor chimeric channels.

TABLE 2 Efficacy of various ligands against selected mutant chimeric ionchannels. EC50-MP EC50-Iss EC50-Ipeak α7 mutant IPD cpd (μM +/− s.e.m.)(μM +/− s.e.m.) (μM +/− s.e.m.) wt 5HT3 ACh 5.4 (1.1) 26 (6)  38 (2) PNU 0.06 (0.02) 0.09 (0.02) 0.8 (0.5) 212 >100 nd nd 241 >100 nd nd245 >100 nd nd W77F 5HT3 28S 3.0 (0.1) 2.4 11.3 34S 1.4 (0.1) nd nd 132S4.3 (0.5) nd nd Q79G 5HT3 ACh 8.2 (2)   9.3 (3.0) 38 (13) 9S 2.3 (0.9)2.3 (0.5) 4.3 (0.9) 22S 0.8 (0.2) 0.4 (0.2) 2.7 (0.9) 38R 1.6 (0.2) ndnd 165S 1.9 (0.4) 0.9 (0.2) 5.9 (0.1) 212 >100 nd nd 241 >100 nd nd245 >100 nd nd Q79G Q139M 5HT3 22S 0.06 (0.04) nd nd 38R 0.23 (0.07) ndnd Q79G Q139G 5HT3 ACh 31  47 (2.0) 296 (64)  22S 3.9 2.7 (1)   11.6(3.6)  Q79G L141S 5HT3 ACh >100 155 (48)  356 (16)  9S 4.1 (0.2)   4(0.2) 12.3 (1.4)  L141F 5HT3 19S 1.4 (0.2) nd nd 88S 0.1 (0.1) nd nd 89S0.4 (0.1) 0.7 (0.2) 3.2 (0.5) 212 0.75 nd nd 241 0.1 nd nd 245 10 nd ndL141F 5HT3 HC ACh nd 18.1 (5.9)  21.6 (3.2)  89S nd 1.5 (0.3) 2.3 (0.3)L141F GlyR ACh nd 190 (120) *complex 89S nd 1.8 (0.4) *complex 118S nd3.0 (0.8) 3.2 (0.4) 119S nd 2.5 (0.4) 3.8 (.9)  L141F Y115F GlyR ACh nd490 (230) 590 (200) 89S nd 5.2 (1.9) 6.9 (1.9) Q79G GlyR 22S nd 4.8(1.2) 7.5 (2.4) L141F GABAC 89S nd 4.9 (3.2) 8.8 (1.4) nd: notdetermined. *complex: multiphasic dose response

To develop these selective LBDs for use in the brain, we also reducedthe responsiveness to the endogenous ligand, ACh. We focused on shiftingEC50_(Ipeak) because steady state concentrations of ACh do not riseabove 100 nM (Vinson and Justice (1997) Journal of Neuroscience Methods,Vol. 73: 61-67), which is significantly below activation threshold ofthis channel. Previously, it was reported for α7 nAChR that mutations atresidues equivalent to α7-5HT3 Y115F and Y210F selectively eliminate AChresponsiveness while leaving nicotine efficacy largely unaffected (Galziet al. (1991) FEBS Letters, Vol. 294: 198-202). Despite the analogouspharmacologic properties of nicotine and aminoquinuclidine benzamides,these mutations greatly reduced or eliminated efficacy of PNU-282987with α7-5HT3. However, inspection of FIG. 1G revealed multiple bindingsite mutations which diminish ACh responsiveness. We reasoned that somecould be combined with selectivity-inducing mutations to generate ligandbinding domains that had orthogonal ligand specificity to the endogenousreceptors while being unresponsive to physiologic levels of ACh. Indeed,α7-5HT3 Q79G Q139G retained responsiveness to 22S (EC50_(Iss)=2.7±1.0μM, EC50_(Ipeak)=11.6±3.6 μM), while for ACh (EC50_(Ipeak)=296±64 μM)the additional Q139G mutation resulted in a nearly 8-fold shift inefficacy from α7-5HT3 Q79G (ACh: EC50_(Ipeak)=38±2 μM). Also, forα7-5HT3 Q79G L141S there was only slight shift in the efficacy for 9S,but ACh responsiveness (EC50_(Ipeak)=356±16 μM) shifted nearly 10-foldwith addition of the L141S mutation (Table 2).

To develop a neuron activating cation channel, we further modified theα7-5HT3 channel with three previously described mutations that werereported to provide a 100-fold increase in conductance: R425Q R429DR433A. Addition of this high conductance (HC) mutation, showed theexpected increase in channel noise upon ligand application (FIG. 7A).After expression of α7-5HT3 HC L141F in layer ⅔ cortical neurons,compound 89S (3 μM) sustainably increased firing rate in these neurons(FIG. 7B).

For neuron silencing, we generated chimeric channels using α7 Q79G or α7L141F fused to the glycine receptor IPD (SEQ ID NO: 8 and 9,respectively). These channels produced large, slowly activating currents(FIG. 8A). To maximize orthogonality with the neuronal activatorsdescribed above, we further developed α7-GlyR L141F (SEQ ID NO: 9). Theligand activated current in cells expressing this channel showedreversal at the chloride reversal potential, indicating that itoperated, expectedly, as a chloride channel (FIG. 8B). A notableproperty is the extremely slow channel activation, which wascharacterized previously for α7-GlyR (Grater et al. (2005) Proc. Natl.Acad. Sci. USA, Vol. 102: 18207-18212). This slow activation is a usefulfeature that can be expected to serve as a low pass filter for unwantedactivation by fast synaptic ACh activity. ACh-responsiveness can befurther diminished by modifying the α7-GlyR L141F with the Y115Fmutation which reduced the already low ACh-responsiveness of thischannel (Table 2).

Layer ⅔ cortical neurons expressing α7-GlyR L141F Y115F show adramatically reduced excitability. This was measured by the amount ofcurrent required to evoke and action potential, also termed rheobase(FIG. 8C). We found no difference in the rheobase of α7-GlyR L141F Y115Fexpressing neurons and untransfected cells. However, in the presence of10 μM compound 89S rheobase increased 10-fold, which demonstrates thestrong reduction of excitability in these neurons. Notably,untransfected cells showed no change in rheobase in the presence ofcompound 89S. Importantly, the effect on neuronal excitability israpidly reversible and when 89S was washed away with drug-free buffer,the neuronal excitability returned to normal.

We also demonstrated this silencing capability using viral gene deliveryvectors with cell type-specific promoters. The synapsin promoter, whichrestricts gene expression to neuronal cell types, was used to driveexpression of α7-GlyR L141F in an adeno-associated virus (AAV) genedelivery vector. Neurons transduced with this virus were renderedsensitive to silencing by compound 89S (FIG. 9A), while untransducedneurons were unaffected by compound 89S (FIG. 9B). These experimentsdemonstrate the capacity to target specific cell populations with thesemutant chimeric ion channels to robustly manipulate electrical activity.

Example 5 Chemical Syntheses

Standard procedures and chemical transformation and related methods arewell known to one skilled in the art, and such methods and procedureshave been described, for example, in standard references such asFiesers' Reagents for Organic Synthesis, John Wiley and Sons, New York,N.Y., 2002; Organic Reactions, vols. 1-83, John Wiley and Sons, NewYork, N.Y., 2006; March J. and Smith M., Advanced Organic Chemistry, 6thed., John Wiley and Sons, New York, N.Y.; and Larock R. C.,Comprehensive Organic Transformations, Wiley-VCH Publishers, New York,1999.

Preparative HPLC was performed using a Varian PrepStar Model SD-1instrument with Agilent prep-C18 preparative column 30×150 mm 10 micron.Detection and collection wavelengths were 240 nm and flowrate was 25ml/min. Solvent A: water with 0.1% TFA, Solvent B: Methanol. Int. 10% B,hold for 5 min at 10% B, 45 min to 60% B.

Example 5A (S)-2-methoxy-N-(quinuclidin-3-yl)benzamide (19S)

1.5 ml of Acetonitrile and Triethylamine (700 uL, 5.0 mmol) was added toa flask charged with (S)-(−)-3-Aminoquinuclidine dihydrochloride (199mg, 1.0 mmol) and o-Methoxybenzoic acid (167 mg, 1.1 mmol), followed byaddition of 2-Chloro-1-methyl pyridinium iodide (383 mg, 1.5 mmol) atroom temperature. After stirring overnight, about 2 ml of water wasadded to reaction mixture to resolve a clear solution that wassubsequently injected into a Preparative HPLC instrument forpurification. Desired fractions were combined and concentrated undervacuum. (S)-2-methoxy-N-(quinuclidin-3-yl)benzamide (221 mg, 85% yield)was isolated as an oil. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.79 (dd,J=7.7 1.6 Hz, 1H), 7.51 (td, J=8.0, 1.6 Hz, 1H), 7.15 (d, J=8.5 Hz, 1H),7.06 (t, J=7.4 Hz, 1H), 4.45 (m, 4H), 3.97 (s, 3H), 3.82 (ddd, J=11.7,9.9, 2.0 Hz 1H), 3.37 (m, 4H), 2.37 (q, J=3.2 Hz, 1H), 2.20 (m, 1H),2.09 (m, 2H), 1.99 (m, 1H). LCMS (ESI): m/z 261.1 (M+H[C₁₅H₂₀N₂O]=261.15).

Example 5B (S)—N-(quinuclidin-3-yl)-4-(trifluoromethyl)benzamide (22S)

1.5 ml of Acetonitrile and Triethylamine (700 uL, 5.0 mmol) was added toa flask charged with (S)-(−)-3-Aminoquinuclidine dihydrochloride (199mg, 1.0 mmol) and α,α,α-Trifluoro-p-toluic acid (209 mg, 1.1 mmol),followed by addition of 2-Chloro-1-methyl pyridinium iodide (383 mg, 1.5mmol) at room temperature. After stirring overnight, about 2 ml of waterwas added to reaction mixture to resolve a clear solution that wassubsequently injected into a Preparative HPLC instrument forpurification. Desired fractions were combined, concentrated undervacuum, and then recrystallized from 4 mL of Methanol/water (3:2) toisolate white crystalline solid of(S)—N-(quinuclidin-3-yl)-4-(trifluoromethyl)benzamide (278 mg, 93%yield). ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 8.05 (d, J=8.2 Hz, 1H),7.82 (d, J=8.2 Hz, 1H), 4.47 (m, 1H), 3.87 (t, J=11.8, 11.8 Hz, 1H),3.38 (m, 4H), 2.39 (m, 1H), 2.27 (m, 2H), 2.13 (t, J=8.2 Hz, 1H). LCMS(ESI): m/z 299.1 (M+H [C₁₅H₁₇F₃N₂O]=299.13).

Example 5C (S)-4-butyl-N-(quinuclidin-3-yl)benzamide (Compound 9S)

1.5 ml of Acetonitrile and Triethylamine (700 uL, 5.0 mmol) was added toa flask charged with (S)-(−)-3-Aminoquinuclidine dihydrochloride (199mg, 1.0 mmol) and 4-Butylbenzoic acid (196 mg, 1.1 mmol), followed byaddition of 2-Chloro-1-methylpyridinium iodide (383 mg, 1.5 mmol) atroom temperature. After stirring overnight, about 2 ml of water wasadded to reaction mixture to resolve a clear solution that wassubsequently injected into a Preparative HPLC instrument forpurification. Desired fractions were combined and concentrated undervacuum. (S)-4-butyl-N-(quinuclidin-3-yl)benzamide (272 mg, 95% yield)was isolated as an oil. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 8.24 (d,J=8.2 Hz, 2H), 8.41 (d, J=8.4 Hz, 2H), 4.42 (m, 1H), 3.84 (ddd, J=11.7,9.9, 2.6 Hz, 1H), 3.35 (m, 4H), 2.69 (t, J=7.7 Hz, 2H), 2.36 (q, J=3.1Hz, 1H), 2.24 (m, 1H), 2.10 (m, 2H), 1.94 (m, 1H), 1.62 (q, J=7.6 Hz,2H), 1.37 (q, J=7.4 Hz, 2H), 0.95 (t, J=7.4 Hz, 3H). LCMS (ESI): m/z287.1 (M+H [C₁₈H₂₆N₂O]=287.20).

Example 5D (S)-5-butyl-N-(quinuclidin-3-yl)pyridine-2-carboxamide(Compound 16S)

1.5 ml of Acetonitrile and Triethylamine (700 uL, 5.0 mmol) was added toa flask charged with (S)-(−)-3-Aminoquinuclidine dihydrochloride (199mg, 1.0 mmol) and Fusaric acid (197 mg, 1.1 mmol), followed by additionof 2-Chloro-1-methylpyridinium iodide (383 mg, 1.5 mmol) at roomtemperature. After stirring overnight, about 2 ml of water was added toreaction mixture to resolve a clear solution that was subsequentlyinjected into a Preparative HPLC instrument for purification. Desiredfractions were combined and concentrated under vacuum.(S)-5-butyl-N-(quinuclidin-3-yl)pyridine-2-carboxamide (273 mg, 95%yield) was isolated as clear brown colored oil. ¹H NMR (400 MHz,Methanol-D4) δ (ppm): 8.52 (d, J=1.8 Hz, 1H), 8.03 (d, J=8.0 Hz, 1H),7.84 (dd, J=8.0 Hz, 1H), 4.50 (m, 1H), 3.82 (ddd, J=10.0, 3.0, 2.6 Hz,1H), 3.49 (m, 1H), 3.37 (m, 3H), 2.76 (t, J=7.8 Hz, 2H), 2.38 (m, J=3.1Hz, 1H), 2.26 (m, 1H), 2.12 (td, J=8.0, 3.1 Hz 2H), 1.96 (m, 1H), 1.68(m, J=7.7 Hz, 2H), 1.40 (m, J=4.5 Hz, 2H), 0.97 (t, J=7.4 Hz, 3H). LCMS(ESI): m/z 288.1 (M+H [C₁₇H₂₅N₃O]=288.20).

Example 5E (S)—N-(quinuclidin-3-yl)-4-(thiophen-2-yl)benzamide (Compound34S)

0.5 ml of Acetonitrile, 0.25 ml of water, and Triethylamine (108 uL,0.78 mmol) was dissolved in a flask charged with(S)-(−)-3-Aminoquinuclidine dihydrochloride (47.8 mg, 0.2 mmol) at roomtemperature. To the mixture was added 4-(2-Thienyl)benzoic acid (49 mg,2.4 mmol) and 1-hydroxy benzotriazole (33.8 mg, 0.25 mmol) followed byaddition of N,N′-Diisopropyl carbodimide (38.7 uL, 0.25 mmol). Themixture was then stirred overnight at room temperature. About 2 ml ofmethanol was added to reaction mixture to resolve a clear solution thatwas subsequently injected into a Preparative HPLC instrument forpurification. Desired fractions were combined and concentrated undervacuum. (S)—N-(quinuclidin-3-yl)-4-(thiophen-2-yl)benzamide (8.2 mg, 13%yield) was isolated at an oil. ¹H NMR (400 MHz, Methanol-D4) δ (ppm):7.90 (dt, J=8.7, 1.9 Hz, 2H), 7.76 (dt, J=8.6, 1.9 Hz, 2H), 7.53 (dd,J=3.7, 1.1 Hz, 1H), 7.48 (dd, J=4.0, 1.1 Hz, 1H), 7.14 (dd, J=3.5, 1.6Hz, 1H), 4.45 (m, 1H), 3.86 (ddd, J=10.0, 3.0, 2.5 Hz, 1H), 3.37 (m,4H), 2.38 (m, 1H), 2.26 (m, 1H), 2.11 (m, 2H), 1.95 (m, 1H). LCMS (ESI):m/z 313.1 (M+H [C₁₈H₂₀N₂OS]=313.13).

Example 5F (S)-2,4-dimethoxy-N-(quinuclidin-3-yl)benzamide (Compound88S)

4.0 ml of DMF, N,N′-Diisopropylethylamine (5.6 ml, 32.14 mmol), and2,4-Dimethoxybenzoic acid (1.75 g, 9.64 mmol) was added to a flaskcharged with (S)-(−)-3-Aminoquinuclidine dihydrochloride (1.60 g, 8.03mmol) and stirred ice-water bath under nitrogen atmosphere. To themixture was added 2-(1H-7-Azabenzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate Methanaminium (3.05 g, 8.03 mmol) thenmixture temperature was raised to ambient temperature slowly by removingthe ice-water bath for 60 minutes. Reaction mixture was worked up byaddition of 1N sodium hydroxide aqueous solution and extracted twicewith ethyl acetate, combined organic layer was washed with saturatedbrine solution, dried over sodium sulfate, filtered, and concentrated tocrude clear oil. Purification via preparative HPLC to obtain pureproduct of (S)-2,4-dimethoxy-N-(quinuclidin-3-yl)benzamide, (1.95 g, 84%yield) ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.42 (m, 1H), 7.11 (s,2H), 4.45 (m, 1H), 3.95 (s, 3H), 3.83 (m, 1H), 3.80 (s, 3H), 3.39-3.27(m, 5H), 2.37 (m, 1H), 2.19 (m, 1H), 2.11 (m, 2H), 2.01 (m, 1H).C16H22N2O3 =290.16 LCMS (M+H): m/z 291

Example 5G (S)-4-methyl-N-(quinuclidin-3-yl)benzamide TFA (Compound176S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 4-methylbenzoic acid in place ofo-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.77 (d,J=8 Hz, 2H), 7.30 (d, J=8 Hz, 2H), 4.44 (m, 1H), 3.82 (m, 1H), 3.41-3.27(m, 5H), 2.40 (s, 3H), 2.35 (m, 1H), 2.24 (m, 1H), 2.09 (m, 2H), 1.92(m, 1H). C₁₅H₂₀N₂O=244.16 LCMS (M+H): m/z 245

Example 5H (S)-4-ethyl-N-(quinuclidin-3-yl)benzamide TFA (Compound 162S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 4-ethylbenzoic acid in place ofo-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.80 (d,J=8 Hz, 2H), 7.34 (d, J=8 Hz, 2H), 4.44 (m, 1H), 3.84 (m, 1H), 3.37-3.27(m, 5H), 2.72 (dd, J=16 Hz, 2H), 2.36 (m, 1H), 2.24 (m, 1H), 2.10 (m,2H), 1.94 (m, 1H), 1.26 (t, J=8 Hz, 3H). C₁₆H₂₂N₂O=258.17 LCMS (M+H):m/z 259

Example 5I (S)-4-propyl-N-(quinuclidin-3-yl)benzamide TFA (Compound165S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 4-propylbenzoic acid in place ofo-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.80 (d,J=8 Hz, 2H), 7.31 (d, J=8 Hz, 2H), 4.44 (m, 1H), 3.83 (m, 1H), 3.41-3.28(m, 5H), 2.66 (t, J=8 Hz, 2H), 2.36 (m, 1H), 2.24 (m, 1H), 2.09 (m, 2H),1.93 (m, 1H), 1.67 (m, 2H), 0.95 (t, J=6 Hz, 3H). C₁₇H₂₄N₂O=272.19 LCMS(M+H): m/z 273

Example 5J (S)-4-pentyl-N-(quinuclidin-3-yl)benzamide TFA (Compound115S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 4-pentylbenzoic acid in place ofo-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.80 (d,J=8 Hz, 2H), 7.31 (d, J=12 Hz, 2H), 4.45 (m, 1H), 3.82 (m, 1H),3.43-3.27 (m, 5H), 2.67 (t, J=8 Hz, 2H), 2.36 (m, 1H), 2.24 (m, 1H),2.09 (m, 2H), 1.92 (m, 1H), 1.64 (m, 2H), 1.34 (m, 2H), 0.90 (t, J=8 Hz,3H). C₁₉H₂₈N₂O=300.22 LCMS (M+H): m/z 301

Example 5K (S)-4-butoxy-N-(quinuclidin-3-yl)benzamide TFA (Compound116S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 4-butoxybenzoic acid in place ofo-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.84 (d,J=8 Hz, 2H), 6.98 (d, J=8 Hz, 2H), 4.43 (m, 1H), 4.04 (t, J=8 Hz, 2H),3.82 (m, 1H), 3.43-3.27 (m, 5H), 2.35 (m, 1H), 2.24 (m, 1H), 2.09 (m,2H), 1.92 (m, 1H), 1.78 (m, 2H), 1.52 (m, 2H), 1.00 (t, J=6 Hz, 3H).C₁₈H₂₆N₂O₂=302.20 LCMS (M+H): m/z 303

Example 5L (S)-4-(pentyloxy)-N-(quinuclidin-3-yl)benzamide TFA (Compound117S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 4-pentyloxybenzoic acid in place ofo-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.84 (d,J=8 Hz, 2H), 6.98 (d, J=8 Hz, 2H), 4.43 (m, 1H), 4.04 (t, J=6 Hz, 2H),3.82 (m, 1H), 3.43-3.27 (m, 5H), 2.35 (m, 1H), 2.24 (m, 1H), 2.09 (m,2H), 1.93 (m, 1H), 1.80 (m, 2H), 1.44 (m, 4H), 0.95 (t, J=6 Hz, 3H).C₁₉H₂₈N₂O₂=316.22 LCMS (M+H): m/z 317

Example 5M (S)-4-isopropyl-N-(quinuclidin-3-yl)benzamide TFA (Compound163S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 4-isopropylbenzoic acid in place ofo-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.80 (d,J=8 Hz, 2H), 7.35 (d, J=8 Hz, 2H), 4.45 (m, 1H), 3.83 (m, 1H), 3.43-3.28(m, 5H), 2.98 (m, 1H), 2.36 (m, 1H), 2.24 (m, 1H), 2.09 (m, 2H), 1.93(m, 1H), 1.27 (d, J=8 Hz, 6H). C₁₇H₂₄N₂O=272.19 LCMS (M+H): m/z 273

Example 5N (S)-4-(dimethylamino)-N-(quinuclidin-3-yl)benzamide TFA(Compound 164S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 4-dimethylaminobenzoic acid in place ofo-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.78 (d,J=8 Hz, 2H), 6.79 (d, J=12 Hz, 2H), 4.41 (m, 1H), 3.82 (m, 1H),3.43-3.36 (m, 4H), 3.25 (m, 1H), 3.05 (s, 6H), 2.35 (m, 1H), 2.25 (m,1H), 2.09 (m, 2H), 1.94 (m, 1H). C₁₆H₂₃N₃O=273.18 LCMS (M+H): m/z 274

Example 5O(S)-2-fluoro-N-(quinuclidin-3-yl)-4-(trifluoromethyl)benzamide TFA(Compound 157S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 2-fluoro-4-(trifluoromethyl)benzoicacid in place of o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ(ppm): 7.85 (t, J=8 Hz, 1H), 7.61 (t, J=8 Hz, 2H), 4.49 (m, 1H), 3.85(m, 1H), 3.37 (m, 4H), 3.26 (ddd, J=4, 13, 6 Hz, 1H), 2.38 (m, 1H), 2.21(m, 1H), 2.11 (td, J=8, 4 Hz, 1H), 1.96 (m, 1H). Ca₅H₁₆F₄N₂O=316.12 LCMS(M+H): m/z 317

Example 5P(R)-2-fluoro-N-(quinuclidin-3-yl)-4-(trifluoromethyl)benzamide TFA(Compound 170R)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 2-fluoro-4-(trifluoromethyl)benzoicacid in place of o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ(ppm): 7.85 (t, J=8 Hz, 1H), 7.61 (t, J=8 Hz, 2H), 4.49 (m, 1H), 3.85(m, 1H), 3.38 (m, 4H), 3.25 (ddd, 1H), 2.38 (m, 1H), 2.21 (m, 1H), 2.11(m, 2H), 1.96 (m, 1H). C₁₅H₁₆F₄N₂O=316.12 LCMS (M+H): m/z 317

Example 5Q(S)-3-fluoro-N-(quinuclidin-3-yl)-4-(trifluoromethyl)benzamide TFA(Compound 156S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 3-fluoro-4-(trifluoromethyl)benzoicacid in place of o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ(ppm): 7.82 (m, 3H), 4.47 (m, 1H), 3.84 (m, 1H), 3.48-3.31 (m, 5H), 2.38(m, 1H), 2.25 (m, 1H), 2.11 (m, 2H), 1.95 (m, 1H). C₁₅H₁₆F₄N₂O=316.12LCMS (M+H): m/z 317

Example 5R(S)—N-(quinuclidin-3-yl)-5-(trifluoromethyl)pyridine-2-carboxamide(Compound 295 S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 5-(trifluoromethyl)picolinic acid inplace of o-methoxybenzoic acid. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 9.06(s, 1H), 8.30 (dd, J=8 Hz, 1H), 7.79 (d, J=8 Hz, 1H), 6.42 (s, 1H), 4.19(m, 1H), 3.47 (dd, J=16 Hz, 1H), 2.92-2.85 (m, 4H), 2.64 (ddd, J=16 Hz,1H), 2.08 (m, 1H), 1.74 (m, 3H), 1.58 (m, 1H). C₁₄H₁₆F₃N₃O=299.12 LCMS(M+H): m/z 300

Example 5S(R)—N-(quinuclidin-3-yl)-5-(trifluoromethyl)pyridine-2-carboxamide(Compound 292 R)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 5-(trifluoromethyl)picolinic acid inplace of o-methoxybenzoic acid. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.98(s, 1H), 8.22 (dd, J=8 Hz, 1H), 7.71 (d, J=8 Hz, 1H), 6.36 (s, 1H), 4.11(m, 1H), 3.39 (dd, J=16 Hz, 1H), 2.83-2.77 (m, 4H), 2.56 (ddd, J=16 Hz,1H), 2.00 (m, 1H), 1.66 (m, 3H), 1.50 (m, 1H). C₁₄H₁₆F₃N₃O=299.12 LCMS(M+H): m/z 300

Example 5T(R)—N-(quinuclidin-3-yl)-6-(trifluoromethyl)pyridine-3-carboxamide(Compound 293 R)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 6-(trifluoromethyl)nicotinic acid inplace of o-methoxybenzoic acid. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.84(s, 1H), 8.34 (dd, J=8 Hz, 1H), 8.23 (s, 1H), 8.12 (dd, J=8 Hz, 1H),4.18 (m, 1H), 3.39 (ddd, J=10, 14 Hz, 1H), 2.95-2.87 (m, 4H), 2.67 (ddd,J=16 Hz, 1H), 2.06 (m, 1H), 1.79-1.74 (m, 3H), 1.50 (m, 1H).C₁₄H₁₆F₃N₃O=299.12 LCMS (M+H): m/z 300

Example 5U(S)—N-(quinuclidin-3-yl)-6-(trifluoromethyl)pyridine-3-carboxamide(Compound 296 S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 6-(trifluoromethyl)nicotinic acid inplace of o-methoxybenzoic acid. ¹H NMR (400 MHz, CDCl₃) S (ppm): 8.84(s, 1H), 8.34 (dd, J=8 Hz, 1H), 8.23 (s, 1H), 8.12 (dd, J=8 Hz, 1H),4.18 (m, 1H), 3.39 (ddd, J=10, 14 Hz, 1H), 2.96-2.87 (m, 4H), 2.68 (ddd,J=16 Hz, 1H), 2.06 (m, 1H), 1.82 (m, 1H), 1.74 (m, 1H), 1.55 (m, 1H).C₁₄H₁₆F₃N₃O=299.12 LCMS (M+H): m/z 300

Example 5V (S)—N-(quinuclidin-3-yl)-4-(trifluoromethoxy)benzamide TFA(Compound 154S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 4-trifluoromethoxybenzoic acid in placeof o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.99(d, J=8 Hz, 2H), 7.35 (d, J=12 Hz, 2H), 4.47 (m, 1H), 3.84 (m, 1H),3.47-3.30 (m, 5H), 2.37 (m, 1H), 2.25 (m, 1H), 2.10 (m, 2H), 1.94 (m,1H). C₁₅H₁₇F₃N₂O₂=314.12 LCMS (M+H): m/z 315

Example 5W (S)—N-(quinuclidin-3-yl)-4-(trifluoromethylthio)benzamide TFA(Compound 149S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 4-(trifluoromethylthio)benzoic acid inplace of o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm):7.97 (d, J=8 Hz, 2H), 7.82 (d, J=8 Hz, 2H), 4.46 (m, 1H), 3.84 (m, 1H),3.42-3.29 (m, 5H), 2.38 (m, 1H), 2.25 (m, 1H), 2.10 (m, 2H), 1.94 (m,1H). C₁₅H₁₇F₃N₂OS=330.10 LCMS (M+H): m/z 331

Example 5X (S)-4-(neopentyloxy)-N-(quinuclidin-3-yl)benzamide TFA(Compound 158S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 4-tert-butoxybenzoic acid in place ofo-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.86 (d,J=12 Hz, 2H), 7.00 (d, J=8 Hz, 2H), 4.43 (m, 1H), 3.82 (m, 1H), 3.69 (s,2H), 3.42-3.29 (m, 5H), 2.35 (m, 1H), 2.24 (m, 1H), 2.08 (m, 2H), 1.92(m, 1H), 1.05 (s, 9H). C₁₉H₂₈N₂O₂=316.22 LCMS (M+H): m/z 317

Example 5Y (R)-4-(methylsulfonyl)-N-(quinuclidin-3-yl)benzamide TFA(Compound 38R)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 34S using 4-(methylsulfonyl)benzoic acid in placeof 4-(2-thienyl)benzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm):8.08 (d, J=4 Hz, 4H), 4.47 (m, 1H), 3.86 (m, 1H), 3.45-3.31 (m, 5H),3.18 (s, 3H), 2.38 (m, 1H), 2.26 (m, 1H), 2.11 (m, 2H), 1.95 (m, 1H).C₁₅H₂₀N₂O₃S=308.12 LCMS (M+H): m/z 309

Example 5Z (S)-4-(methylsulfonyl)-N-(quinuclidin-3-yl)benzamide TFA(Compound 134S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 4-(methylsulfonyl)benzoic acid in placeof o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 8.08(d, J=4 Hz, 4H), 4.47 (m, 1H), 3.86 (m, 1H), 3.42-3.30 (m, 5H), 3.18 (s,3H), 2.39 (m, 1H), 2.26 (m, 1H), 2.11 (m, 2H), 1.96 (m, 1H).C₁₅H₂₀N₂O₃S=308.12 LCMS (M+H): m/z 309

Example 5AA (S)-3-(methylsulfonyl)-N-(quinuclidin-3-yl)benzamide TFA(Compound 148S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 3-(methylsulfonyl)benzoic acid in placeof o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 8.44(t, 1H), 8.21 (dt, J=8 Hz, 1H), 8.15 (dt, J=8 Hz, 1H), 7.77 (t, J=8 Hz,1H), 4.48 (m, 1H), 3.86 (m, 1H), 3.44-3.30 (m, 5H), 3.18 (s, 3H), 2.40(m, 1H), 2.27 (m, 1H), 2.11 (m, 2H), 1.95 (m, 1H). C₁₅H₂₀N₂O₃S=308.12LCMS (M+H): m/z 309

Example 5AB (S)-2-propoxy-N-(quinuclidin-3-yl)benzamide TFA (Compound208S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using o-propoxybenzoic acid in place ofo-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.81 (dd,J=8 Hz, 1H), 7.50 (m, 1H), 7.15 (d, J=8 Hz, 1H), 7.05 (td, J=6 Hz, 1H),4.45 (m, 1H), 4.13 (m, 2H), 3.87 (m, 1H), 3.39 (m, 4H), 3.22 (m, 1H),2.35 (m, 1H), 2.20 (m, 1H), 2.11 (m, 2H), 2.01 (m, 1H), 1.91 (m, 2H),1.08 (t, J=6 Hz 3H). C₁₇H₂₄N₂O₂=288.18 LCMS (M+H): m/z 289

Example 5AC (S)-2-methoxy-4-methyl-N-(quinuclidin-3-yl)benzamide TFA(Compound 86S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 2-methoxy-4-methylbenzoic acid in placeof o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.75(d, J=8 Hz, 1H), 7.00 (s, 1H), 6.90 (d, J=8 Hz, 1H), 4.44 (m, 1H), 3.98(s, 3H), 3.38-3.39 (m, 5H), 2.41 (s, 3H), 2.37 (m, 1H), 2.19 (m, 1H),2.10 (m, 2H), 2.00 (m, 1H). C₁₆H₂₂N₂O₂=274.17 LCMS (M+H): m/z 275

Example 5AD (S)-2,5-dimethoxy-N-(quinuclidin-3-yl)benzamide (Compound89S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 88S using 2,5-dimethoxybenzoic acid in place of2,4-dimethoxybenzoic acid. ¹H NMR (400 MHz, Acetone-D4) δ (ppm): 7.61(m, J=4 Hz, 1H), 7.11 (s, 1H), 7.07 (m, J=4 Hz, 1H), 4.07 (m, 1H), 4.00(s, 3H), 3.80 (s, 3H), 3.34 (s, 3H), 2.89-2.82 (m, 4H), 2.62 (m, 1H),1.98 (m, 1H), 1.84 (m, 1H), 1.84 (m, 2H), 1.52 (m, 1H).C₁₆H₂₂N₂O₃=290.16 LCMS (M+H): m/z 291

Example 5AE (S)-4-chloro-2-methoxy-N-(quinuclidin-3-yl)benzamide(Compound 90S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 34S using 4-chloro-2-methoxybenzoic acid in placeof 4-(2-thienyl)benzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm):7.75 (d, J=8 Hz, 1H), 7.21 (s, 1H), 7.09 (dd, J=8 Hz, 1H), 4.44 (m, 1H),3.98 (s, 3H), 3.80 (m, 3H), 3.38-3.27 (m, 3H), 2.37 (m, 1H), 2.18 (m,1H), 2.10 (m, 2H), 1.98 (m, 1H). C₁₆H₂₂N₂O₂=294.11 LCMS (M+H): m/z 295

Example 5AF (S)-2-phenoxy-N-(quinuclidin-3-yl)benzamide TFA (Compound91S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 34S using 2-phenoxybenzoic acid in place of4-(2-thienyl)benzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.74(dd, J=8 Hz, 1H), 7.51 (m, 1H), 7.38 (m, 1H), 7.28 (td, J=8 Hz, 1H),7.15 (t, J=8 Hz, 1H), 7.01 (m, 1H), 4.33 (m, 1H), 3.74 (m, 3H), 3.29 (m,3H), 3.16 (m, 1H), 2.97 (m, 1H), 2.19 (m, 1H), 2.02 (m, 3H), 1.82 (m,1H). C₂₀H₂₂N₂O₂=322.17 LCMS (M+H): m/z 323

Example 5AG (S)-5-chloro-2-methoxy-N-(quinuclidin-3-yl)benzamide TFA(Compound 118S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 5-chloro-2-methoxybenzoic acid in placeof o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.74(d, J=4 Hz, 1H), 7.49 (dd, J=8 Hz, 1H), 7.16 (d, J=12 Hz, 1H), 4.33 (m,1H), 3.97 (s, 3H), 3.82 (m, 1H), 3.38 (m, 4H), 3.27 (m, 1H), 2.37 (m,1H), 2.18 (m, 1H), 2.10 (m, 3H), 1.99 (m, 1H). C₁₅H₁₉ClN₂O₂=294.11 LCMS(M+H): m/z 295

Example 5AH (S)-5-fluoro-2-methoxy-N-(quinuclidin-3-yl)benzamide TFA(Compound 119S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 5-fluoro-2-methoxybenzoic acid in placeof o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.54(m, 1H), 7.27 (m, 1H), 7.17 (m, 1H), 4.45 (m, 1H), 3.97 (s, 3H), 3.83(m, 1H), 3.39 (m, 4H), 3.27 (m, 1H), 2.38 (m, 1H), 2.19 (m, 1H), 2.10(m, 2H), 1.99 (m, 1H). C₁₅H₁₉FN₂O₂=278.14 LCMS (M+H): m/z 279

Example 5AI (S)-2-methoxy-5-methyl-N-(quinuclidin-3-yl)benzamide TFA(Compound 120S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 2-methoxy-5-methylbenzoic acid in placeof o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.62(m, 1H), 7.32 (m, 1H), 7.04 (d, J=4 Hz, 1H), 4.44 (m, 1H), 3.94 (s, 3H),3.82 (m, 1H), 3.38-3.27 (m, 5H), 2.36 (m, 1H), 2.31 (s, 3H), 2.19 (m,1H), 2.10 (m, 2H), 1.99 (m, 1H). C₁₆H₂₂N₂O₂=274.17 LCMS (M+H): m/z 275

Example 5AJ (S)-2,4,5-trimethoxy-N-(quinuclidin-3-yl)benzamide TFA(Compound 121S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 2,4,5-trimethoxybenzoic acid in placeof o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.50(s, 1H), 6.76 (s, 1H), 4.44 (m, 1H), 4.02 (s, 3H), 3.93 (s, 3H), 3.82(s, 3H, m, 1H), 3.40-3.28 (m, 5H), 2.36 (m, 1H), 2.17 (m, 1H), 2.10 (m,2H), 2.01 (m, 1H). C₁₇H₂₄N₂O₄=320.17 LCMS (M+H): m/z 321

Example 5AK (S)—N-(quinuclidin-3-yl)-2-(trifluoromethoxy)benzamide TFA(Compound 122S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 2-(trifluoromethoxy)benzoic acid inplace of o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm):7.64 (m, 2H), 7.48 (td, J=8 Hz 1H), 7.42 (m, 1H), 4.48 (m, 1H), 3.85 (m,1H), 3.37 (m, 4H), 3.17 (m, 1H), 2.33 (m, 1H), 2.20 (m, 1H), 2.10 (m,2H), 1.95 (m, 1H). C₁₇H₂₄F₃N₂O₂=314.12 LCMS (M+H): m/z 315

Example 5AL (S)-3-methoxy-N-(quinuclidin-3-yl)naphthalene-2-carboxamideTFA (Compound 127S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 3-methoxy-2-naphthoic acid in place ofo-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 8.26 (s,1H), 7.85 (d, 2H), 7.53 (m, 1H), 7.40 (m, 1H), 4.49 (m, 1H), 4.04 (s,3H), 3.84 (m, 1H), 3.39-3.29 (m, 5H), 2.40 (m, 1H), 2.23 (m, 1H), 2.10(m, 2H), 1.99 (m, 1H). C₁₉H₂₂N₂O₂=310.17 LCMS (M+H): m/z 311

Example 5AM (S)-2,4,6-trimethoxy-N-(quinuclidin-3-yl)benzamide TFA(Compound 211S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 2,4,6-trimethoxy benzoic acid in placeof o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 6.24(s, 1H), 4.13 (m, 1H), 3.83 (s, 3H), 3.80 (s, 6H), 3.38 (m, 1H), 2.94(m, 4H), 2.76 (m, 1H), 2.12 (m, 1H), 2.00 (m, 1H), 1.82 (m, 2H), 1.59(m, 1H). C₁₇H₂₄N₂O₄=320.17 LCMS (M+H): m/z 321

Example 5AN (S)-2-methoxy-N-(quinuclidin-3-yl)pyridine-3-carboxamide(Compound 294S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 2-methoxynicotinic acid in place ofo-methoxybenzoic acid. ¹H NMR (400 MHz, CDCl₃) δ (ppm): 8.51 (dd, J=8 Hz1H), 8.28 (dd, J=6 Hz 1H), 7.07 (dd, J=8 Hz 1H), 4.16 (s, 3H, m, 1H),3.44 (dd, J=12 Hz, 1H), 2.92 (t, J=8 Hz 2H), 2.85 (m, 2H), 2.61 (ddd,J=16, 12 Hz, 1H), 2.03 (m, 1H), 1.75 (m, 3H), 1.59 (m, 1H).C₁₄H₁₉N₃O₂=261.15 LCMS (M+H): m/z 262

Example 5AO (S)-2-ethoxy-N-(quinuclidin-3-yl)benzamide TFA (Compound85S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using o-ethoxybenzoic acid in place ofo-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.85 (dd,J=8 Hz, 1H), 7.50 (m, 1H), 7.12 (d, J=8 Hz, 1H), 7.85 (t, 1H), 4.45 (m,1H), 4.23 (m, 2H), 3.87 (m, 1H), 3.41 (m, 4H), 3.25 (m, 1H), 2.37 (m,1H), 2.22 (m, 1H), 2.11 (m, 2H), 2.02 (m, 1H), 1.51 (m, J=6 Hz, 3H).C₁₆H₂₂N₂O₂=274.17 LCMS (M+H): m/z 275

Example 5AP (S)—N-(quinuclidin-3-yl)biphenyl-4-carboxamide TFA (Compound28S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using biphenyl-4-carboxylic acid in place ofo-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.86 (d,J=8 Hz, 2H), 7.63 (d, J=12 Hz, 2H), 7.57 (d, J=8 Hz, 2H), 7.37 (t, J=8Hz, 2H), 7.29 (t, J=8 Hz, 1H), 4.38 (m, 1H), 3.75 (m, 1H), 3.36 (m, 1H),3.26 (m, 4H), 2.28 (m, 1H), 2.17 (m, 1H), 2.00 (m, 2H), 1.84 (m, 1H).C₂₀H₂₂N₂O=306.17 LCMS (M+H): m/z 307

Example 5AQ (R)-4′-propyl-N-(quinuclidin-3-yl)biphenyl-4-carboxamide TFA(Compound 96R)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 4′-propylbiphenyl-4-carboxylic acid inplace of o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm):7.80 (d, J=8 Hz, 2H), 7.73 (d, J=8 Hz, 2H), 7.59 (d, J=8 Hz, 2H), 7.29(d, J=8 Hz, 2H), 4.47 (m, 1H), 3.86 (m, 1H), 3.45-3.30 (m, 5H), 2.64 (t,J=8 Hz, 2H), 2.39 (m, 1H), 2.27 (m, 1H), 2.11 (m, 2H), 1.95 (m, 1H),1.69 (m, 2H), 0.97 (d, J=8 Hz, 3H). C₂₃H₂₈N₂O=348.22 LCMS (M+H): m/z 349

Example 5AR (S)-4′-propyl-N-(quinuclidin-3-yl)biphenyl-4-carboxamide TFA(Compound 131S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 96. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.93(d, J=8 Hz, 2H), 7.73 (d, J=8 Hz, 2H), 7.59 (d, J=8 Hz, 2H), 7.29 (d,J=8 Hz, 2H), 4.47 (m, 1H), 3.86 (m, 1H), 3.45-3.30 (m, 5H), 2.64 (t, J=8Hz, 2H), 2.39 (m, 1H), 2.27 (m, 1H), 2.11 (m, 2H), 1.95 (m, 1H), 1.69(m, 2H), 0.97 (d, J=8 Hz, 3H). C₂₃H₂₈N₂O=348.22 LCMS (M+H): m/z 349

Example 5AS (R)-4′-fluoro-N-(quinuclidin-3-yl)biphenyl-4-carboxamide TFA(Compound 97R)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 19S, using 4′-fluorobiphenyl-4-carboxylic acid inplace of o-methoxybenzoic acid. ¹H NMR (400 MHz, Methanol-D4) δ (ppm):7.95 (d, J=8 Hz, 2H), 7.70 (m, 4H), 7.21 (d, J=8 Hz, 2H), 4.47 (m, 1H),3.86 (m, 1H), 3.45-3.31 (m, 5H), 2.39 (m, 1H), 2.27 (m, 1H), 2.11 (m,2H), 1.95 (m, 1H). C₂₀H₂₁FN₂O=324.16 LCMS (M+H): m/z 325

Example 5AT (S)-4′-fluoro-N-(quinuclidin-3-yl)biphenyl-4-carboxamide TFA(Compound 132S)

Title compound was synthesized according to the procedure used in thesynthesis of Compound 97. ¹H NMR (400 MHz, Methanol-D4) δ (ppm): 7.95(d, J=8 Hz, 2H), 7.71 (m, 4H), 7.21 (d, J=6 Hz, 2H), 4.47 (m, 1H), 3.86(m, 1H), 3.45-3.30 (m, 5H), 2.39 (m, 1H), 2.28 (m, 1H), 2.12 (m, 2H),1.96 (m, 1H). C₂₀H₂₁FN₂O=324.16 LCMS (M+H): m/z 325

Example 5AU (S)-4-iodo-N-(quinuclidin-3-yl)benzamide (Compound 273S)

N,N′-Diisopropylethylamine (1.05 ml, 6.03 mmol) was added to a flaskcharged with DMF solution mixture of (S)-(−)-3-Aminoquinuclidinedihydrochloride (400 mg, 2.01 mmol) and 4-Iodobenzoyl chloride (616 mg,2.31 mmol). The clear yellow solution was stirred for 3 hours. Thereaction mixture was worked up by addition of 1N sodium hydroxideaqueous solution and extracted twice with ethyl acetate, combinedorganic layer was washed with saturated brine solution, dried oversodium sulfate, filtered, and concentrated to crude clear oil. The crudeoil was purified via preparative HPLC to obtain pure product of(S)-4-iodo-N-(quinuclidin-3-yl)benzamide, (460 mg, 64% yield). ¹H NMR(400 MHz, Methanol-D4) δ (ppm): 7.85 (d, J=8 Hz, 2H), 7.60 (d, J=8 Hz,2H), 4.12 (m, 1H), 3.31 (m, 1H), 2.99 (m, 1H), 2.84 (m, 4H), 2.02 (m,1H), 1.90 (m, 1H), 1.78 (m, 2H), 1.54 (m, 1H). C₁₄H₁₇IN₂O=356.04 LCMS(M+H): m/z 357

Example 5AV(S)—N-(quinuclidin-3-yl)-4′-(trifluoromethyl)biphenyl-4-carboxamide(Compound 274S)

4-(trifluoromethyl)phenylboronic acid (20.9 mg, 0.11 mmol) was added toa flask charged with (S)-4-iodo-N-(quinuclidin-3-yl)benzamide (35.6 mg,0.1 mmol), cesium carbonate (81.5 mg, 0.25 mmol) in 2 ml of Acetonitrileand 2 ml of water. The mixture was degassed by sonication for 20 minutesfollowed by charging catalytic amount of Palladium (II) acetate andplace under nitrogen to stirred for 3 hours. The reaction mixture wasfiltered through pad of celite and washed with acetone, solution wasconcentrated under vacuum to oil. Preparative HPLC instrument was usedto obtain pure(S)—N-(quinuclidin-3-yl)-4′-(trifluoromethyl)biphenyl-4-carboxamide(24.5 mg, 65% yield). ¹H NMR (400 MHz, CDCl₃) δ (ppm): 7.80 (d, J=8 Hz,2H), 7.63 (m, 4H), 7.58 (d, J=8 Hz, 2H), 6.33 (d, 1H), 4.11 (m, 1H),3.38 (m, 1H), 2.80 (m, 4H), 2.56 (ddd, J=6, 14, 6 Hz, 1H), 2.00 (m, 1H),1.65 (m, 3H), 1.47 (m, 1H). C₂₁H₂₁F₃N₂O=374.16 LCMS (M+H): m/z 375

Example 5AW (S)-3′-fluoro-N-(quinuclidin-3-yl)biphenyl-4-carboxamide(Compound 278S)

Title compound was synthesized according to the procedure used in thesynthesis of compound 274, using 3-(fluoromethyl)phenylboronic acid inplace of 4-(trifluoromethyl)phenylboronic acid. ¹H NMR (400 MHz, CDCl₃)δ (ppm): 7.77 (d, J=8 Hz, 2H), 7.55 (d, J=8 Hz, 2H), 7.32 (m, 2H), 7.21(m, 1H), 7.00 (m, 1H), 6.33 (d, 1H), 4.09 (m, 1H), 3.37 (m, 1H), 2.83(m, 4H), 2.54 (m, 1H), 1.99 (m, 1H), 1.65 (m, 3H), 1.47 (m, 1H).C₂H₂₁FN₂O=324.16 LCMS (M+H): m/z 325

Example 5AX (S)-2′-fluoro-N-(quinuclidin-3-yl)biphenyl-4-carboxamide(Compound 279S)

Title compound was synthesized according to the procedure used in thesynthesis of compound 274, using 3-(fluoromethyl)phenylboronic acid inplace of 4-(trifluoromethyl)phenylboronic acid. ¹H NMR (400 MHz, CDCl₃)δ (ppm): 7.77 (d, J=8 Hz, 2H), 7.53 (dd, J=8 Hz, 2H), 7.32 (td, J=8 Hz,1H), 7.28 (m, 1H), 7.12 (m, 2H), 6.34 (d, 1H), 4.08 (m, 1H), 3.37 (m,1H), 2.793 (m, 4H), 2.53 (m, 1H), 1.98 (m, 1H), 1.64 (m, 3H), 1.44 (m,1H). C₂H₂₁FN₂O=324.16 LCMS (M+H): m/z 325

Example 5AY(S)—N-(quinuclidin-3-yl)-3′-(trifluoromethyl)biphenyl-4-carboxamide(Compound 281S)

Title compound was synthesized according to the procedure used in thesynthesis of compound 274, using 3-(trifluoromethyl)phenylboronic inplace of 4-(trifluoromethyl)phenylboronic acid. ¹H NMR (400 MHz, CDCl₃)δ (ppm): 7.87 (d, J=8 Hz, 2H), 7.80 (s, 1H), 7.74 (d, J=8 Hz, 1H), 7.62(m, 3H), 7.56 (t, J=8 Hz, 1H), 6.56 (d, 1H), 4.16 (m, 1H), 3.42 (m, 1H),2.85 (m, 4H), 2.56 (m, 1H), 2.06 (m, 1H), 1.79 (m, 3H), 1.53 (m, 1H).C₂₁H₂₁F₃N₂O=374.16 LCMS (M+H): m/z 375

Example 5AZ Compound 212

A catalytic amount of Palladium (II) acetate (10.9 mg, 0.05 mmol) wasadded to microwave reaction vial charged with2-Methylene-3-quinuclidinone hydrochloride (174.4 mg, 1.00 mmol),Phenylhydrazine (109 mg, 1.01 mmol) in 2 ml of acetic acid. Sealed thevial with a cap then heated to 150° C. for 15 minutes. Added 1.0M K₂CO₃and extracted with ethyl acetate 2×50 ml, combined organic layer waswashed with brine solution, dried over sodium sulfate, filtered, andconcentrated under vacuum. Purified by flash chromatography 0% to 10%methanol and 2% TEA in CH₂Cl₂ to recover white solid Compound 212 (12.1mg, 5% yield) ¹H NMR (400 MHz, CDCl₃) β (ppm): 7.19 (s, 1H), 7.07 (t,J=8 Hz, 2H), 6.85 (td, J=7 Hz, 1H), 6.7 (d, J=8 Hz, 1H), 3.60 (d, J=12Hz, 1H), 3.29 (dd, J=14 Hz, 1H), 3.00 (m, 4H), 2.82 (m, 1H), 2.50 (m,1H). C₁₄H₁₇N₃=277.14 LCMS (M+H): m/z 278

Compounds related to Compound 212 can be prepared according to thepathways identified in Scheme 1.

Example 5BA Compound 301

A solution of (2-bromophenyl)methanamine, 2-Methylene-3-quinuclidinonehydrochloride, and 1,4-Diazabicyclo[2.2.2]octane in DMF, is degassed viasonication for 20 minutes then a catalytic amount of Palladium (II)acetate is charged under nitrogen and stir at reflux to give Compound301.

Example 5BB Compound 302

Compound 301 is reduced with sodium borohydride in THF solution to yieldCompound 302.

Example 5BC Compound 303

Compound 302 is oxidized with a solution of KMnO₄ and MnO₂ in DCM andstirred at room temperature to yield Compound 303.

Example 5BD Compound 305

Compound 302 is treated with cyanoborohydride, formaldehyde, in aceticacid to yield Compound 305.

It is understood that the disclosed invention is not limited to theparticular methodology, protocols and reagents described as these mayvary. It is also understood that the terminology used herein is for thepurposes of describing particular embodiments only and is not intendedto limit the scope of the present invention which will be limited onlyby the appended claims.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of skill in the artto which the disclosed invention belongs. Although any methods andmaterials similar or equivalent to those described herein can be used inthe practice or testing of the present invention, the exemplary methods,devices, and materials are as described. All patents, patentapplications and other publications cited herein and the materials forwhich they are cited are specifically incorporated by reference in theirentireties.

Those skilled in the art will recognize, or be able to ascertain usingno more than routine experimentation, many equivalents to the specificembodiments of the invention described herein. Such equivalents areintended to be encompassed by the following claims.

1. A method of modulating the excitability of a neuronal cellcomprising: expressing in the neuronal cell a genetic construct encodinga chimeric receptor, wherein the chimeric receptor comprises a ligandbinding domain from an α7 nicotinic acetylcholine receptor fused to atransmembrane domain from a ligand-gated ion channel protein, whereinsaid ligand binding domain comprises at least one mutation that confersselective binding to a compound; and exposing the neuronal cell to thecompound.
 2. The method of claim 1, wherein the ligand-gated ion channelprotein is selected from the group consisting of ionotropic nicotinicacetylcholine receptors, ionotropic serotonin receptors, ionotropicglycine receptors, and ionotropic GABA receptors.
 3. The method of claim2, wherein the ligand-gated ion channel protein is selective forcations.
 4. The method of claim 3, wherein the ligand-gated ion channelprotein is a 5HT3 receptor.
 5. The method of claim 3, wherein theexcitability of the neuron is increased.
 6. The method of claim 2,wherein the ligand-gated ion channel protein is selective for anions. 7.The method of claim 6, wherein the ligand-gated ion channel protein is aglycine receptor or GABA receptor.
 8. The method of claim 6, wherein theexcitability of the neuron is decreased.
 9. The method of claim 1,wherein the at least one mutation in the ligand binding domain isselected from the group consisting of Q79A, Q79G, L141A, L141F, L141P,W77F, W77Y, and W77M in SEQ ID NO:
 1. 10. The method of claim 1, whereinthe at least one mutation in the ligand binding domain is selected fromthe group consisting of Q79A, Q79G, L141A, L141F, L141P, W77F, W77Y, andW77M in SEQ ID NO:
 6. 11. The method of claim 1, wherein the at leastone mutation in the ligand binding domain is selected from the groupconsisting of Q79A, Q79G, L141A, L141F, L141P, W77F, W77Y, and W77M inSEQ ID NO:
 10. 12. The method of claim 1, wherein the compound does notactive a wild-type α7 nicotinic acetylcholine receptor.
 13. The methodof claim 12, wherein the compound is selected from the group consistingof 9S, 16S, 19S, 22S, 28S, 34S, 38R, 85S, 86S, 88S, 89S, 90S, 91S, 96R,97R, 115S, 117S, 118S, 119S, 120S, 121S, 127S, 131S, 132S, 134S, 148S,149S, 154S, 156S, 157S, 158S, 163S, 164S, 165S, 170R, 208S, 212, 241,242, 245, 253, 254, 255, 278S, 279S, 281S, 292R, 294S, 295S, and 296S.14. The method of claim 1, wherein the neuronal cell is in vitro. 15.The method of claim 1, wherein the neuronal cell is in vivo.
 16. Achimeric receptor comprising a ligand binding domain from an α7nicotinic acetylcholine receptor fused to a transmembrane domain from aligand-gated ion channel protein, wherein the ligand binding domaincomprises at least one mutation that confers selective binding to acompound.
 17. The chimeric receptor of claim 16, wherein thetransmembrane domain is a transmembrane domain of a ligand-gated ionchannel protein selected from the group consisting of ionotropicnicotinic acetylcholine receptors, ionotropic serotonin receptors,ionotropic glycine receptors, and ionotropic GABA receptors.
 18. Thechimeric receptor of claim 17, wherein the transmembrane domain is thetransmembrane domain from a 5HT3 receptor, the transmembrane domain of aglycine receptor, or a transmembrane domain of a GABA C receptor. 19.The chimeric receptor of claim 18, wherein the at least one mutation inthe ligand binding domain is W77F in SEQ ID NO: 1, SEQ ID NO: 6, or SEQID NO:
 10. 20. The chimeric receptor of claim 19, wherein the chimericreceptor selectively binds a compound selected from the group consistingof 28S, 34S, 96R, 97R, 131S, 132S, 278S, 279S, and 281S.
 21. Thechimeric receptor of claim 18, wherein the at least one mutation in theligand binding domain is Q79A or Q79G in SEQ ID NO: 1, SEQ ID NO: 6, orSEQ ID NO:
 10. 22. The chimeric receptor of claim 21, wherein thechimeric receptor selectively binds a compound selected from the groupconsisting of 9S, 16S, 22S, 38R, 115S, 117S, 134S, 148S, 149S, 154S,1565, 157S, 158S, 163S, 164S, 165S, 170R, 292R, 295S, and 296S.
 23. Thechimeric receptor of claim 18, wherein the at least one mutation in theligand binding domain is L141F or L141P in SEQ ID NO: 1, SEQ ID NO: 6,or SEQ ID NO:
 10. 24. The chimeric receptor of claim 23, wherein thechimeric receptor selectively binds a compound selected from the groupconsisting of 19S, 85S, 86S, 88S, 89S, 90S, 91S, 118S, 119S, 120S, 121S,127S, 208S, 212, 241, 242, 245, 253, 254, 255, and 294S.
 25. Thechimeric receptor of claim 18, wherein the chimeric receptor has theamino acid sequence of SEQ ID NO: 2, SEQ ID NO: 3, SEQ ID NO: 4, SEQ IDNO: 5, SEQ ID NO: 7, SEQ ID NO: 8, SEQ ID NO: 9, SEQ ID NO: 11, SEQ IDNO: 12, or SEQ ID NO:
 13. 26. A kit comprising a chimeric receptor ofclaim 16 and at least one compound.
 27. The kit of claim 26, wherein theat least one mutation in the ligand binding domain of the chimericreceptor is W77F in SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO:
 10. 28.The kit of claim 27, wherein the compound is 28S, 34S, 96R, 97R, 131S,132S, 278S, 279S, and/or 281S.
 29. The kit of claim 26, wherein the atleast one mutation in the ligand binding domain of the chimeric receptoris Q79A or Q79G in SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO:
 10. 30. Thekit of claim 29, wherein the compound is 9S, 16S, 22S, 38R, 115S, 117S,134S, 148S, 149S, 154S, 156S, 157S, 158S, 163S, 164S, 165S, 170R, 292R,2955, and/or
 2965. 31. The kit of claim 26, wherein the at least onemutation in the ligand binding domain of the chimeric receptor is L141For L141P in SEQ ID NO: 1, SEQ ID NO: 6, or SEQ ID NO:
 10. 32. The kit ofclaim 31, wherein the compound is 19S, 85S, 86S, 88S, 89S, 90S, 91S,118S, 119S, 120S, 121S, 127S, 208S, 212, 241, 242, 245, 253, 254, 255,and/or 294S.
 33. A method of treating a disease or disorder associatedwith the nervous system in a subject in need thereof comprising:delivering a genetic construct to a population of neurons in thesubject, wherein the genetic construct encodes a chimeric receptor,wherein the chimeric receptor comprises a ligand binding domain from anα7 nicotinic acetylcholine receptor fused to a transmembrane domain froma ligand-gated ion channel protein, wherein said ligand binding domaincomprises at least one mutation that confers selective binding to acompound; and administering the compound to the subject.
 34. The methodof claim 33, wherein the compound is capable of passing through theblood brain barrier.
 35. The method of claim 33, wherein the activity ofthe population of neurons is increased following administration of thecompound to the subject.
 36. The method of claim 33, wherein theactivity of the population of neurons is decreased followingadministration of the compound to the subject.
 37. The method of claim33, wherein the disorder is epilepsy and the population of neuronscomprises neurons in the seizure focus.
 38. The method of claim 33,wherein the disorder is chronic pain and the population of neurons areC-fiber neurons in the dorsal root ganglia.
 39. A method of treating adisease or disorder associated with the nervous system in a subject inneed thereof comprising: delivering a genetic construct to a populationof neurons in the subject, wherein the genetic construct encodes achimeric receptor, wherein the chimeric receptor comprises a ligandbinding domain from an α7 nicotinic acetylcholine receptor fused to atransmembrane domain from a ligand-gated ion channel protein, whereinsaid ligand binding domain comprises at least one mutation that confersselective binding to a compound of formula I, and administering saidcompound of formula Ito said subject, wherein said compound of formula Iis represented by the following structure:

or a pharmaceutically acceptable salt thereof, wherein: A is one of:

wherein, each of R₁, R₄ and R₅ is independently selected from the groupof hydrogen, halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆ thioalkyl,C₁-C₆haloalkyl, C₁-C₆ thiohaloalkyl, C₁-C₆haloalkoxy, amino, alkylamino,dialkylamino, alkylsulfonyl, aryl and heteroaryl, wherein said aryl andheteroaryl are optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆alkoxy, C₁-C₆ thioalkyl, C₁-C₆haloalkyl, C₁-C₆ thiohaloalkyl, C₁-C₆haloalkoxy, amino, alkylamino, dialkylamino or alkylsulfonyl; each of R₂and R₃ is independently selected from the group of hydrogen, halo, C₁-C₆alkyl, C₁-C₆ alkoxy, C₁-C₆ thioalkyl, C₁-C₆haloalkyl, C₁-C₆thiohaloalkyl, C₁-C₆ haloalkoxy, amino, alkylamino, dialkylamino,alkylsulfonyl, aryl and heteroaryl, wherein said aryl and heteroaryl areoptionally substituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₁-C₆thioalkyl, C₁-C₆haloalkyl, C₁-C₆ thiohaloalkyl, C₁-C₆ haloalkoxy, amino,alkylamino, dialkylamino or alkylsulfonyl; or R₂ and R₃, together withthe carbon atoms to which they are attached, form a 5 or 6-memberedcarbocyclic or heterocyclic ring optionally substituted with halo, C₁-C₆alkyl, C₁-C₆ alkoxy, C₁-C₆ thioalkyl, C₁-C₆haloalkyl, C₁-C₆thiohaloalkyl, C₁-C₆haloalkoxy, amino, alkylamino, dialkylamino,alkylsulfonyl; with the provisos that: (a) at least one of R₁, R₂, R₃,R₄ and R₅ is not hydrogen; and (b) if neither of R₁ and R₅ is C₁-C₆alkoxy, then R₃ is present and is not hydrogen.
 40. A method accordingto claim 39, wherein said compound of formula I is the (S)-enantiomer.41. A method according to claim 39, wherein said at least one mutationin said ligand binding domain is W77F in SEQ ID NO: 1, SEQ ID NO: 6 orSEQ ID NO:
 10. 42. A method according to claim 39, wherein said at leastone mutation in said ligand binding domain is Q79G or Q79A in SEQ ID NO:1, SEQ ID NO: 6 or SEQ ID NO:
 10. 43. A method according to claim 39,wherein said at least one mutation in said ligand binding domain isL141F or L141P in SEQ ID NO: 1, SEQ ID NO: 6 or SEQ ID NO:
 10. 44. Amethod according to claim 39, wherein A is

wherein R₃ is phenyl optionally substituted with halo, C₁-C₆ alkyl,C₁-C₆ alkoxy or C₁-C₆ haloalkyl.
 45. A method according to claim 44,wherein each of R₁, R₂, R₄ and R₅ is hydrogen.
 46. A method according toclaim 45, wherein said at least one mutation in said ligand bindingdomain is W77F in SEQ ID NO:1, SEQ ID NO:6 or SEQ ID NO:10.
 47. A methodaccording to claim 39, wherein A is

wherein R₃ is C₃-C₆ alkyl, C₅-C₆ alkoxy, C₁-C₂ haloalkoxy, C₁-C₂thiohaloalkyl, C₁-C₂ perhaloalkyl, dialkylamino or alkylsulfonyl.
 48. Amethod according to claim 47, wherein A is

wherein each of R₁, R₂, R₄ and R₅ is hydrogen.
 49. A method according toclaim 48, wherein said at least one mutation in said ligand bindingdomain is Q79G or Q79A in SEQ ID NO: 1, SEQ ID NO: 6 or SEQ ID NO: 10.50. A method according to claim 47, wherein A is

wherein R₃ is C₁-C₂ perhaloalkyl or C₃-C₆ alkyl.
 51. A method accordingto claim 50, wherein: each of R₄ and R₅ is hydrogen; R₁, if present, ishydrogen; and R₂, if present, is hydrogen.
 52. A method according toclaim 50, wherein said at least one mutation in said ligand bindingdomain is Q79G or Q79A in SEQ ID NO: 1, SEQ ID NO: 6 or SEQ ID NO: 10.53. A method according to claim 39, wherein: at least one of R₁ and R₅is methoxy, ethoxy or phenoxy.
 54. A method according to claim 53,wherein A is


55. A method according to claim 54, wherein: R₁ is methoxy, ethoxy orphenoxy; each of R₂, R₃ and R₄ is independently hydrogen, chloro,fluoro, methoxy, ethoxy, methyl or ethyl; and R₅ is hydrogen.
 56. Amethod according to claim 53, wherein said at least one mutation in saidligand binding domain is L141F or L141P in SEQ ID NO: 1, SEQ ID NO: 6 orSEQ ID NO:
 10. 57. A method of treating a disease or disorder associatedwith the nervous system in a subject in need thereof comprising:delivering a genetic construct to a population of neurons in thesubject, wherein the genetic construct encodes a chimeric receptor,wherein the chimeric receptor comprises a ligand binding domain from anα7 nicotinic acetylcholine receptor fused to a transmembrane domain froma ligand-gated ion channel protein, wherein said ligand binding domaincomprises at least one mutation that confers selective binding to acompound of formula II, and administering said compound of formula II tosaid subject, wherein said compound of formula II is represented by thefollowing structure:

or a pharmaceutically acceptable salt thereof, wherein: A is aryl orheteroaryl optionally substituted with halo, C₁-C₆ alkyl, C₁-C₆ alkoxy,C₁-C₆ thioalkyl, C₁-C₆ haloalkyl, furanyl or thiophenyl, wherein saidfuranyl and thiophenyl are optionally substituted with halo, C₁-C₆alkyl, C₁-C₆ alkoxy, C₁-C₆thioalkyl or C₁-C₆ haloalkyl; each of R^(A)and R^(B) is hydrogen or C₁-C₆ alkyl; or R^(A) and R^(B) taken togetherare ═O; and n is 0 or
 1. 58. A method according to claim 57, wherein: Ais phenyl, pyridyl, pyrazinyl or quinoxalinyl, wherein said phenyl,pyridyl, pyrazinyl or quinoxalinyl is substituted with halo, C₁-C₆alkyl, C₁-C₆ alkoxy, C₁-C₆thioalkyl or C₁-C₆ haloalkyl.
 59. A methodaccording to claim 57, wherein said at least one mutation in said ligandbinding domain is L141F or L141P in SEQ ID NO: 1, SEQ ID NO: 6 or SEQ IDNO:
 10. 60. A compound of formula III:

wherein each

is a single or double bond; each R^(A) and R^(B) is hydrogen or R^(A)and R^(B) taken together are ═O; E is —N, —NH, CR^(C), or —CR^(C)R^(D),each R^(C) and R^(D) is hydrogen or R^(C) and R^(D) taken together are═O; or a pharmaceutically acceptable salt thereof; wherein said compounddoes not comprise adjacent double bonds.
 61. The compound of claim 60,wherein said compound is selected from the group consisting of:


62. The compound of claim 61 having the structure:

or a pharmaceutically acceptable salt thereof.